CN104487568A - Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof - Google Patents

Abstract

The disclosure provided herein relates generally to mesenchymal -like stem cells 'hES-T-MiSC' or 'T-MSC' and the method of producing the stem cells. The method comprises culturing embryonic stem cells under conditions that the embryonic stem cells develop through an intermediate differentiation of trophoblasts, and culturing the differentiated trophoblasts to hES-T-MSC or T-MSC, T-MSC derived cells and cell lineages 'T-MSC-DL' are also described. Disclosed also herein are solutions and pharmaceutica! compositions comprising the T-MSC and/or T-MSC-DL, methods of making the T-MSC and T-MSC-DL, and methods of using the T-MSC and T-MSC-DL for treatment and prevention of diseases, specifically, T-MSC and T-MSC-DL are used as immunosuppressive agents to treat multiple sclerosis and autoimmune diseases.

Description

The mesenchymal stem cells of derived from human embryonic stem, method and application thereof

The reference of related application

The right of priority of the right of priority of the U.S. Provisional Patent Application 61/670,192 of application claims submission on July 11st, 2012 and the U.S. Provisional Patent Application 61/684,509 of submission on August 17th, 2012.Be incorporated herein by reference with its entirety at this.

1. introduction

Disclosure provided herein generally relates to the method for mesenchymal stem cells " hES-T-MSC " or " T-MSC " and the described stem cell of preparation, described method comprises cultivates embryonic stem cell under the following conditions: embryonic stem cell is grown through the differentiation of intermediate product nurse cell, and is broken up to hES-T-MSC or T-MSC by nurse cell.Content disclosed herein is T-MSC, solution, pharmaceutical composition (comprising T-MSC), prepare T-MSC method, use T-MSC treatment and prophylactic method, particularly, use T-MSC as immunosuppressant treatment multiple sclerosis and other autoimmune disorder, be applied to tissue regeneration and/or reparation, and use described T-MSC through hemato encephalic barrier and blood spinal cord barrier with the method for delivering drugs.Content disclosed herein also comprises and uses T-MSCs immunity moderation system, suppress the immune response of individual autoantigen and repair impaired central nervous system.Composition disclosed herein comprises for immunoregulatory T-MSCs, because the T-MSC of the modification providing immunosuppression capability to be improved by the expression of modifying factor.

2. background technology

Human mesenchymal stem cell/stroma cell has been widely used in immune system and tissue repair.Human embryo stem cell can be used as source reliably and produces high-quality human mesenchymal stem cell to induce.Had multiple method inducing human embryo stem cell to break up to mesenchymal cell at present, but method used at present can not obtain high yield, highly purified mescenchymal stem cell with high-level efficiency induction.

Mescenchymal stem cell (mesenchymal stem cells, MSC), a kind of adult stem cell deriving from the tissue such as adult mouse and human marrow, umbilical cord fat, there is multi-lineage potential, this stem cell under given conditions can induced development lipoblast, chondrocyte, scleroblast, neurocyte, sarcoplast, the mature cell such as stroma cell and inoblast.These inductive technologies are very ripe and be subject to the protection of patent.For example, referring to U.S. Patent number 5,486,359 (human mesenchymal stem cells).

But the mescenchymal stem cell in current available adult tissue source also exists some defects.The first, as limited in derived from bone marrow and the quality of donor tissue differs, and limits the investigation and application of MSCs, and the stdn hindering MSCs to use as pharmaceutical prod larger scale clinical.The second, the MSCs deriving from adult tissue is the cell mass of high complexity, only has wherein a part of cell to have powerful immunosuppression capability.Clinical application needs a large amount of MSCs, usually needs to carry out amplification in vitro MSCs, but the immunity of amplification in vitro decline MSCs is resisted and ability of going back to the nest (Javazon et al., 2004).3rd, clinical application adult origin MSCs exists the propagation of safety problem as vicious transformation (Wong, 2011) and the potential infectious agent of donor.

For overcoming above defect, scientist attempts, by different methods, hESCs is induced to differentiate into MSCs, comprising hESCs and mouse bone marrow OP9 Dual culture, use cell multiplex Summing Factor chemical reagent to induce and manually select (Barberi et al., 2005; Chen et al., 2012; Liu et al., 2012; Sanchez et al., 2011).Recent research report, TGF signal β pathway inhibitor SB431542 is applied to induction hESCs and is divided into MSCs, the method simplifies induction flow process and improves induced efficiency (Chen et al.,, but the output of MSCs and purity all very low (see following controlled trial) 2012).In 2010, the inventor of Advanced Cell Technology, Inc. developed another kind of method, and induction angioblast is divided into MSCs, although the method uses cytokine and the methylcellulose medium of many costlinesses, induced efficiency is still very low.

HESCs induction known today is that the multiple method of MSCs has one or more shortcomings and weakness.Wherein, the induction method of hESCs and OP9 co-culture of cells has length consuming time, yields poorly, purity is low, use the shortcomings (Barberi et al., 2005) such as the feeder layer cells of animal and indefinite culture condition; Form the method for breaking up hypertrophy cell around embryoid body from the hESCs of heavy bed board, there is length consuming time, cell yield is low, culture condition is indefinite and the shortcoming (Olivier et al., 2006) such as both expensive; From cultivate hESCs differentiation method on glue primordial covering flat board have yield poorly, the shortcoming (Liu et al., 2012) such as the indefinite and length consuming time of culture condition; Utilize the differentiation method of TGF signal β pathway inhibitor process hESCs have purity low, yield poorly, length consuming time and the low inferior shortcoming of MSCs immunosuppression capability (Chen et al., 2012 that obtain; Sanchez et al., 2011).Therefore, in the urgent need to unrestrictedly, in fact complete, high stability, high induced efficiency are treated with the MSCs in identical source and prevent various disease.

Multiple sclerosis (Multiple sclerosis, MS) be a kind of chronic autoimmune disorder, to be peripheral immunocyte penetrate into central nervous system by the hemato encephalic barrier of damage or blood spinal cord barrier to pathogenic factor, causes the myelin around neural axon to be inflamed and demyelination and aixs cylinder formation scar (McFarland and Martin (2007)).Add up according to American National multiple sclerosis association, the medicine of current U.S. food and FAD approval treatment multiple sclerosis is more than 70 kinds, comprise A Wonasi (Avonex, IFN β-1a), doubly safe dragon (Betaseron, IFN β-1b), FTY720 (Gilenya, sphingosine 1-phosphate acceptors instrumentality), acetic acid copaxone (Glatirameracetate or Copolymer 1), natalizumab (Tysabri, humanized α-alphab-integrin antibodies).But, these medicines only can delay the state of an illness and produce severe side effect, comprise and infect enhancing, heart attack, apoplexy, PML, irregular pulse, pain, depression, fatigue, macular edema and erective dysfunction (Johnstonand So (2012); Weber et al. (2012)).

Because mescenchymal stem cell has function (Auletta etc., (2012) of immunomodulatory and neurotization; Pittenger etc., (1999)) and repair potential (Chao etc., (2009) of hemato encephalic barrier; Menge etc., (2012)), therefore transplanting MSCs treats multipleization sclerosis becomes a kind of potential, very attractive therapy.MSCs has versatility, can induce and produce various kinds of cell system, comprise adipocyte, chondrocyte, scleroblast and neurone.MSCs is present in the tissues such as embryo, the amnion of newborn infant and adult, umbilical cord, marrow and fat.Compared with following with current medicine, MSCs has the advantage of multiple uniqueness, such as, they can be contacted with each other by secretion and cell as carrier that is multiple and the potential Synergistic treatment factor and the tissue migrating to damage and play local effect (Uccelli and Prockop (2010a).Importantly, MSCs has been proved to be able to treat mice with experimental autoimmune encephalomyelitis, and this is a kind of generally acknowledged multiple sclerosis disease mouse model (Gordon etc., 2008a; Gordon etc., (2010); Morando etc., (2012); Peron etc., (2012); Zappia etc., (2005); Zhang etc., (2005)), meanwhile, MSCs is also applied to clinical trial (Connick etc., (2012) of multiple sclerosis; Karussis etc., (2010); Mohyeddin Bonab etc., (2007); Yamout etc., (2010)).Due to mouse and human bone marrow-derived mescenchymal stem cell (bone marrow-derivedMSC, BM-MSC) effectively can both slow down mice with experimental autoimmune encephalomyelitis to worsen, therefore, MSCs xenotransplantation is not problem (Gordon etc., (2008a); Gordon etc., (2010); Morando etc., (2012); Peron etc., (2012); Zappia etc., (2005); Zhang etc., (2005)).But BM-MSC treats mouse EAE model curative effect in different reports and to differ (Gordon etc., (2008a); Payne etc., (2012); Zappia etc., (2005); Zhang etc., (2005)), and then cause the suspection of BM-MSC being treated to MS effect.

Therefore, in the urgent need to unrestricted, real complete, high stability, high induced efficiency and the MSCs treatment of originating consistent and prevention MS and Other diseases.Disclose herein and be divided into hES-T-MSCs by one efficient method induction hES, the method meets above requirement.The content also disclosed herein is analyzed by microarray analysis and other, the different expression patterns at multiple key factor that qualification hES-T-MSC and BM-MSC compares, and hES is divided into other method of MSCs.

3. summary of the invention

A kind of method being derived mesenchymal stem cells by nurse cell induction intermediate steps by hESCs is disclosed herein.The mescenchymal stem cell obtained by this method is called " hES-T-MSC " or " T-MSC ".T-MSC can be divided into various kinds of cell or the clones such as lipocyte, sarcoplast, neuronal cell, scleroblast, inoblast, chondrocyte, mesenchymal cell after induction.Cell or the clone of T-MSC differentiation are referred to as " pedigree that T-MSC is derivative " or " T-MSC-DL ".

Disclosed herein is the composition comprising T-MSC and/or T-MSC-DL, they all have immunosuppressant performance.Disclosed herein is based on replying and T-MSC and/or the T-MSC-DL cell mass of selection by immunity moderation, and there is the composition of immunity moderation performance.Also disclose herein, compared with BM-MSC, there is T-MSC and/or T-MSC-DL of stronger immunosuppression capability.

A kind of method that effectively can obtain the T-MSC of high purity and high yield is disclosed herein.Compared with former report method, described method needs less step and less differentiation factor.

End user's embryonic stem cell (hESCs) derives mesenchymal stem cells method by the intermediate differentiation of nurse cell is disclosed herein.The MSC of described trophoblastic origin is called " hES-T-MSC " or " T-MSC ".This T-MSC is applied to immunity moderation system.Such as, they can bring out the damage of central nervous system by checking immunocyte, thus treat multiple sclerosis efficiently.

The mescenchymal stem cell derived by the Human embryo that openly method is produced herein is disclosed herein.

The method that induction T-MSC is divided into T-MSC-DL is disclosed herein.

Also disclose application T-MSC and/or T-MSC-DL herein and treat the multiple sclerosis of Mammals particularly people experimenter with other human autoimmune's property disease.

Another object of disclosure invention is to provide a kind of cellular product T-MSC to be applied to immunomodulatory.Such as, in tissue or organ transplantation, prevention or Immunosuppression repel.Another specific embodiment of described method lowers or Immunosuppression response, and described immunne response is graft versus host disease (GVH disease).In another specific embodiment, described immunne response is autoimmune disorder, such as diabetes, lupus erythematosus or rheumatic arthritis.

The further object of disclosure invention is to provide the cellular product T-MSC-DL being used for the treatment of nervous system disorders.

Described method can adopt abundant mescenchymal stem cell provided herein in immunne response, produce detectable restraining effect.Such as, the stem cell multiple provided herein for contacting panimmunity cell can comprise 1 × 10 ⁵t-MSC, 1 × 10 ⁶t-MSC, 1 × 10 ⁷t-MSC, 1 × 10 ⁸t-MSC or more.

In one embodiment, methods described herein are a kind of from hESCs derivative (here also referring to produce) MSCs novel method.The method comprises the following steps:

A. human embryo stem cell is cultivated in the serum free medium containing at least one cytokine, and the enough inducing embryo stem cells of cytokine amount contained by this substratum break up to nurse cell.In one embodiment, the cycle being divided into nurse cell needs about 2-5 days.In one embodiment, in substratum, comprise BMP4, simultaneously in TGF beta inhibitor (namely SB431542, A83-01 or ALK5 inhibitor etc.) presence or absence situation, to improve differentiation efficiency.

B. add at least one somatomedin to containing in the culture of nurse cell, and continue to cultivate in serum-free culture, the amount of wherein said somatomedin enough increases nurse cell.In one embodiment, substratum contains BMP4 (this step is optional).

C. nurse cell is separated, then this cell is spread again on the flat board of gelatin, ln, fibronectin, collagen or matrigel bag quilt, and cultivate at the substratum contained or not containing serum, the amount of this substratum enough promotes nurse cell by front-T-MSC to T-MSC cytodifferentiation.In one embodiment, the nurse cell of separation is cultivated after 4-10 days and is produced T-MSC cell, wherein at least about 90%, 95%, 96%, 97%, 98%, 99% cell expressing adult between fill the surface markers of stem cell.In one embodiment, LIF, bFGF or PDGF is comprised in substratum to improve amplification efficiency.

In a concrete embodiment, the nurse cell of derived from human embryonic stem expresses Trop-2, but does not express CD73.

In a concrete embodiment, front-T-MSC expresses Trop-2 and/or CD73.

In a concrete embodiment, described T-MSC expresses CD73, CD105 and CD90.An object of disclosure method is that induction hESCs is divided into highly purified MSCs.In one preferred embodiment, CD73 ⁺, CD105 ⁺and CD90 ⁺t-MSC purity higher than 90%, 95%, 96%, 97%, 98% and 99%.

Find by detecting, the surface markers of a high proportion of T-MSC cell expressing adult mesenchymal cell, proves that T-MSC has very high purity.No matter in vivo or external, the mescenchymal stem cell that described mescenchymal stem cell obtains than other method has higher immunosuppression capability.The mescenchymal stem cell that current open method derives is named as human embryo stem cell-nurse cell-derivative mescenchymal stem cell (hES-trophoblast-derived MSCs) and more succinct is referred to as T-MSC.

In some embodiments, the described blood serum medium that contains comprises foetal calf serum or people AB serum, and adds glutamine.Described serum free medium contains removes serum substitute (knockout serum replacement, KOSR) or bovine serum albumin (bovine serum albumin, BSA).

In some embodiments, there is the step that extra, the T-MSC that this step uses the gamma Rays do not waited from 1gy to 200gy to obtain.

In another embodiment of the present invention, described method can produce and increase and obtain at least 10000 T-MSC, at least 50000 T-MSC, at least 100000 T-MSC, at least 500,000 T-MSC, at least 1 × 10 ⁶individual T-MSC, at least 5 × 10 ⁶individual T-MSC, at least 1 × 10 ⁷individual T-MSC, at least 5 × 10 ⁷individual T-MSC, at least 1 × 10 ⁸individual T-MSC, at least 5 × 10 ⁸individual T-MSC, at least 1 × 10 ⁹individual T-MSC, at least 5 × 10 ⁹t-MSC or at least 1 × 10 ¹⁰t-MSC.These methods can obtain 10000 to 100 hundred million T-MSC cells.In some embodiments, one or more of hES-MSC difference mark is expressed at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Human embryo-mescenchymal stem cell.In some embodiments, described mark is CD73, CD90 and CD105.

In one embodiment, described T-MSCs significance ground reduces the disease score of EAE mouse, and the demyelination that simultaneous reduces, T cell are infiltrated and microglial reaction.In addition, compare with derived from bone marrow mescenchymal stem cell (bone marrow derived MSCs, BM-MSC) with external in vivo, T-MSCs has stronger immunosuppressive activity.Additionally provide the key protein matter/molecule of differential expression between T-MSC and BM-MSC.The expression level qualification that there is provided herein by measuring protein/molecule marker has the method for the T-MSCs of the immunosuppressive activity of improvement.Additionally provide genetic modification to improve the method for the immunosuppressive activity of T-MSCs.

Another embodiment of the invention is a kind of solution comprising T-MSC, and T-MSC at least comprises at least 10000 T-MSC, at least 50000 T-MSC, at least 100000 T-MSC, at least 500000 T-MSC, at least 1 × 10 ⁶individual T-MSC, at least 5 × 10 ⁶individual T-MSC, at least 1 × 10 ⁷individual T-MSC, at least 5 × 10 ⁷individual T-MSC, at least 1 × 10 ⁸individual T-MSC, at least 5 × 10 ⁸individual T-MSC, at least 1 × 10 ⁹individual T-MSC, at least 5 × 10 ⁹t-MSC or at least 1 × 10 ¹⁰t-MSC.

In some embodiments, at least 10000 cells are comprised in the culture of 2ml volume, at least 100000 cells are comprised in the culture of 10ml volume, at least 1000000 cells are comprised in the culture of 100ml volume, comprise at least 10000000 cells in the culture of 1000ml volume, and reach 5 × 10 in 4000ml substratum ⁸individual cell.

These solution can be injected to experimenter.Can these solution frozen.These solution for the manufacture of one-tenth medicament, can be applied to and carry out disease therapy by T-MSC administration.

Present invention also offers a kind of method that production can be applicable to the T-MSC solution be injected in patient body, said method comprising the steps of: the method isolated cell solution utilizing earlier paragraphs to describe, then cell is seeded to and can be applicable to being injected in the liquid of patient.Present invention also offers a kind of method of producing applicable freezing T-MSC solution, the method includes the steps of: utilize the method isolated cell solution described by earlier paragraphs, is then seeded to by cell and can be applicable in freezing solution.

Further, another embodiment of the invention is the T-MSC expressing one or more cell surface marker albumen or its combination, and described labelled protein comprises CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and CD166.In another embodiment, the mescenchymal stem cell of derived from human embryonic stem do not express or the one or more cellular labeled proteins of low expression level or its combination, described mark comprises CD34, CD31, CD45.In another embodiment, the mescenchymal stem cell of derived from human embryonic stem is not expressed or the one or more proinflammatory disease protein labeling of low expression level, or the combination of these short inflammatory proteins, described short inflammatory protein comprises MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and VCAM1.In some embodiments, compared with mesenchymal stem cells MSCs, the mescenchymal stem cell of derived from human embryonic stem expresses the level of above-mentioned mark at least half.

Another embodiment of the invention is a kind of cell culture comprising T-MSC, and wherein T-MSC expresses one or more cellular labeled proteins, comprises CD73, CD90, CD105, CD13, CD29, CD54, CD144, CD146 and CD44.In another embodiment, the T-MSC in cell culture does not express or one or more cellular labeled proteins of low expression level, comprises CD34, CD31 and CD45.In another embodiment, the T-MSC in cell culture does not express or one or more short inflammatory protein of low expression level, comprises MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and VCAM1.

In some embodiments, at least 1 × 10 are comprised in described cell culture ⁶individual T-MSC, at least 1 × 10 ⁷individual T-MSC, at least 1 × 10 ⁸individual T-MSC, at least 1 × 109 T-MSC, or at least 1 × 10 ¹⁰individual T-MSC.

In other embodiment, in cell culture, express CD73 albumen at least about the T-MSC of 90%; At least express CD73 albumen more than the T-MSC of 90%; T-MSC at least about 95% expresses CD73 albumen; Or the T-MSC more than 95% expresses CD73 albumen.In other embodiments, in cell culture, express CD73 albumen at least about the T-MSC of 96%; At least express CD73 albumen more than the T-MSC of 97%; T-MSC at least about 98% expresses CD73 albumen; At least express CD73 albumen more than the T-MSC of 99%.

In other embodiment, in cell culture, express at least about the T-MSC of 75%, 80%, 85%, 90%, 5%, 99% the cell mark albumen that at least one is selected from CD90, CD105, CD44 and CD29.

In other embodiment, do not express at least about the T-MSC of 80%, 85%, 90%, 95% and 99% or low expression level at least one cellular labeled proteins in cell culture, comprise CD34, CD31 and CD45.

In other embodiment, do not express at least about the T-MSC of 75%, 80%, 85%, 90%, 95%, 99% or low expression level at least one proinflammatory protein in cell culture, comprise MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and VCAM1.In some embodiments, described T-MSC expresses high-caliber CD24, TGF β 2 or both high level expressions.

In some embodiments, gamma Rays T-MSC of the present invention or cell culture is used.

Other embodiments of the present invention are pharmaceutical preparations, and its pharmaceutical formulations comprises any one T-MSC described herein or cell culture and pharmaceutically acceptable carrier.

Further, the embodiment of other of the present invention is the frozen preparation of any T-MSC described herein or cell culture.

Also provide a kind of for there being the method for subject and the prevention autoimmune disorder relevant to T cell needing its treatment herein, the method comprises the following steps: to there being its solution comprising T-MSC described in previous paragraph of experimenter's administering therapeutic significant quantity, cell culture or pharmaceutical preparation of needs.The autoimmune disorder relevant to T cell includes but not limited to Crohn's disease, inflammatory bowel, graft versus host disease, systemic lupus erythematous and rheumatic arthritis, also has the delayed hypersensitivity (belonging to the 4th type anaphylaxis) of T cell mediation, i.e. type 1 diabetes, MS, RA, Hashimoto thyroiditis, Crohn's disease, contact dermatitis and scleroderma, etc.

In some embodiments, preferred experimenter is Mammals or birds, the most preferably mankind.In some embodiments, described solution, cell culture and pharmaceutical preparation comprise by radiation or the T-MSC without radiation.

In some embodiments, be used for the treatment of described in comprise with prophylactic method and be used for the treatment of the combination therapy with prophylactic agent with one or more.

In some other embodiments, the present invention is for there being a kind of method needing its experimenter to provide treatment and prevention multiple sclerosis, and the method comprises the following steps: describe the solution of T-MSC, cell culture or pharmaceutical preparation to there being the earlier paragraphs that comprises needing its experimenter to give to treat significant quantity.Described multiple sclerosis can be recurrence/persistent form multiple sclerosis, progress/relapsive sclerosis, primary multiple sclerosis or Secondary cases multiple sclerosis.Experimenter's preferred mammal, and most preferably be the mankind.Described solution, cell culture or pharmaceutical preparation can comprise radiation or the T-MSC without radiation.

Described method comprises and gives experimenter other healing potion further, and this medicament includes but not limited to FTY720, corticotropin (ACTH), methylprednisolone, dexamethasone, IFN β-1a, IFN-1b, acetic acid copaxone, endoxan, methotrexate, azathioprine, CldAdo, S-Neoral, mitoxantrone and sulfasalazine.Further, in another one embodiment, in order to healing potion is delivered to central nervous system, one or more healing potions can be connected on T-MSCs so that through hemato encephalic barrier and/or blood spinal cord barrier.

A kind of method of sending medicament through hemato encephalic barrier and/or blood spinal cord barrier is provided herein, the method comprises the following steps: connected by medicament or be conjugated to T-MSC, then the experimenter he-MSC and agent complexes that need it is had, wherein said T-MSC can through hemato encephalic barrier and/or blood spinal cord barrier, and the i or I having the experimenter needing it can be treated, prevents or be diagnosed to described medicament.T-MSC can be the form of individual cells, cell culture, solution or pharmaceutical preparation.Described medicament can include but not limited to medicine, protein, DNA, RNA and small-molecule substance.

Another embodiment is the delivery system comprising T-MSC and the medicament puted together or be connected, and the object of this system is through hemato encephalic barrier and/or blood spinal cord barrier.

Method described herein has many advantages.An object of the disclosure method makes hESCs pass through the middle nurse cell stage to realize differentiation, this method and existing method different, and have following advantage.

Thering is provided a kind of herein selects clinical rank T-MSC to treat the method for autoimmune disorder, and described T-MSC has following characteristics: (i) cell expressing group-1 mark containing >95%; (ii) cell expressing group-2 mark containing >80%; (iii) cell expressing group-3 mark containing <5%; (iv) IL-10 and TGF β is expressed; (v) cell expressing IL-6, IL-12 and TNF α containing <2%; (vi) express high-caliber CXCR7, CXCL2 and CXCL12, but express the gene of low-level HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family; (vii) all groups-4 marks of the cell coexpression containing <0.001%.Wherein group-1 mark is: CD73, CD90, CD105, CD146, CD166 and CD44.Group-2 mark is: CD13, CD29, CD54 and CD49E.Group-3 mark is: CD45, CD34, CD31 and SSEA4.Group-4 mark is: OCT4, NANOG, TRA-1-60 and SSEA4.

The method of the mescenchymal stem cell providing a kind of T-MSC production a group of modification to modify herein, the mescenchymal stem cell of described modification has following characteristics: (i) cell expressing group-1 mark containing >95%; (ii) cell expressing group-2 mark containing >80%; (iii) cell expressing group-3 mark containing <5%; (iv) IL-10 and TGF β is expressed; (v) cell expressing IL-6, IL-12 and TNF α containing <2%; (vi) containing the mark of cell coexpression all groups-4 being less than 0.001%.Wherein group-1 mark is: CD73, CD90, CD105, CD146, CD166 and CD44.Group-2 mark is CD13, CD29, CD54 and CD49E.Group-3 mark is CD45, CD34, CD31 and SSEA4.Group-4 mark is: OCT4, NANOG, TRA-1-60 and SSEA4.

There is provided conditioned medium, conditioned medium concentrated solution, cell lysate or other its derived prods herein, it comprises one or more biomolecules of being secreted by described T-MSC.

There is provided herein and use T-MSC as feeder cell with the method for increase marrow hemopoietic stem cells and umbilical cord hemopoietic stem cell.In some embodiments, these T-MSC being applicable to open method express Stro3.In some embodiments, T-MSC and marrow hemopoietic stem cells and/or umbilical cord hemopoietic stem cell Dual culture.In some embodiments, T-MSC is mescenchymal stem cell.This provide a kind of coculture be made up of described T-MSC and marrow hemopoietic stem cells.This provide a kind of coculture be made up of described T-MSC and umbilical cord hemopoietic stem cell.

Also disclose the test kit comprising T-MSC described herein.In some embodiments, described test kit comprises T-MSC and cell delivery vehicle.

On the one hand, there is provided herein a kind of suppression or reduce immunoreactive method, the method comprises for some time that multiple immunocyte and multiple T-MSC to be contacted with each other, enough T-MSC can detect ground Immunosuppression reaction during this period of time, wherein, in mixed lymphocyte reacion test, T-MSC can detect propagation and/or the differentiation of ground suppressor T cell.In another particular embodiment of the invention, cell contacts with each other and carries out in vitro.In one more specifically embodiment, the cell in body contacts with each other and to carry out in Mammals is as the mankind.In another particular embodiment of the invention, described cell contacts with each other and comprises intravenously, intramuscular gives T-MSC or be injected into experimenter's organ (as pancreas).

There is provided herein and produce the method for cell mass, described cell mass comprises T-MSC, the selection of these T-MSC is had to the ability of regulation and control (as suppression) immunne response based on them.Such as, in one embodiment, the invention provides a kind of method selecting T-MSC cell mass, this method comprises: (a) analyzes multiple T-MSC in mixed lymphocyte reacion (MLR) test; If b () multiple T-MSC can suppress the propagation of the CD4 positive or CD8 positive T cell in mixed lymphocyte reacion test with detecting, select multiple T-MSC.T-MSC described here expresses CD73, CD90, CD105, CD13, CD29, CD54 and CD 44.In one embodiment, T-MSC does not express or expresses low-level CD34, CD31 and CD45.In one embodiment, T-MSC does not express or expresses low-level MMP2, RAGE, IFNGR2, IL-12A, IL-6 and VCAM1.

There is provided herein the method that T-MSC is divided into other clone multiple, described clone includes but not limited to adipocyte, sarcoplast, neuronal cell line, scleroblast, inoblast, chondrocyte and mesenchymal cell.

There is provided herein use the cellular products of T-MSC and differentiation thereof carry out tissue regeneration and or the method for tissue repair, these methods comprise: give other derivative clones of T-MSC and/or T-MSC of q.s to promote tissue regeneration.Tissue is raw includes but not limited to the regeneration of regeneration of joints, tendon, connective tissue regeneration, neuronal cell line regeneration, adipose tissue regeneration, osteanagenesis, skin regeneration, anathrepsis, regenerating bone or cartilage, unstriated muscle regeneration, Myocardial Regeneration, epithelium regeneration and ligament regeneration.

In a particular embodiment, the ratio of T cell and T-MSC in mixed lymphocyte reacion test, such as about 20:1,15:1,10:1,5:1,2:2,1:1,1:2,1:5,1:10 or 1:20, preferred 10:1.

Another object of disclosure method is that the method for logical a kind of high yield realizes producing a large amount of mescenchymal stem cells efficiently.Disclosure method can produce and obtain being the mescenchymal stem cell of initial hESCs cell count about 10 times.In the process that hESCs is broken up by nurse cell, have few cell loss, but other method there is in initial differentiation step the initiator cell more than 90% to lose, thus causes obtaining the cell concentration more less than disclosure method.

An object of disclosure method is to provide a kind of method can producing mescenchymal stem cell in relative short time.The whole requirements of process completing methods described herein is no more than 6-14 days, and this determines according to initial hES system.

An object of disclosure method is to provide a kind of method of low cost.Described differentiation method only needs very small amount of substratum, only needs a kind of cytokine-BMP4, and BMP4 is very low at the working concentration of present method.

An object of disclosure method is to provide a kind of method of low cost.Differentiation method as herein described only needs very small amount of substratum, only needs a kind of cytokine-BMP4 and/or TGF beta inhibitor (as SB431542, A83-01 or ALK5 inhibitor etc.).

An object of disclosure method is to provide a kind of method of high yield.Differentiation method described here can from 1 × 10 in 30 days ⁵1-5 × 10 are produced in individual hESC ¹⁰individual T-MSC cell, but other method only can be produced and reached 1 × 10 in 30 days ⁸individual MSC.

The further object of disclosure method is to provide the mescenchymal stem cell with high immunosuppression ability.Described T-MSC has higher immunosuppression capability than marrow (BM) and other tissue-derived mescenchymal stem cell.Described T-MSC has higher immunosuppression capability than human embryo stem cell by the mescenchymal stem cell that other method derives.

In a particular embodiment, compared with testing with the mixed lymphocyte reacion not containing T-MSC, the mixed lymphocyte reacion test containing T-MSC suppresses the propagation of at least 50%, 70%, 90% or the 95%CD4 positive or CD8 positive T cell.

In another particular embodiment of the invention, above-mentioned any one composition comprises matrix.In one more specifically embodiment, described matrix is three-dimensional support.In another more specifically embodiment, described matrix comprises collagen, gelatin, ln, fibronectin, pectin, ornithine or vitronectin.In another more specifically embodiment, described matrix is biomaterial.In another more specifically embodiment, described matrix comprises cell outer membrane protein matter.In another more specifically embodiment, described matrix comprises synthetic compound.In another more specifically embodiment, described matrix comprises biologically active substance.In another more specifically embodiment, described biologically active substance is somatomedin, cytokine, antibody or be less than 5000 daltonian organic molecules.

The present invention further provides the population of stem cells of freezen protective, as comprised the cell mass of T-MSC, wherein, described cell mass has immunoregulation ability as described herein.Such as, the invention provides a group can suppress to detectability T propagation and/or differentiation in mixed lymphocyte reacion (MLR) test T-MSC through qualification, wherein, described cell freezen protective, and wherein said cell mass is installed in container.

In a concrete embodiment, the container preserving the cell mass of above-mentioned any one freezen protective is sack.In various embodiment, described cell mass comprises about, at least or maximum 1 × 10 ⁶individual described stem cell, 5 × 10 ⁶individual described stem cell, 1 × 10 ⁷individual described stem cell, 5 × 10 ⁷individual described stem cell, 1 × 10 ⁸individual described stem cell, 5 × 10 ⁸individual described stem cell, 1 × 10 ⁹individual described stem cell, 5 × 10 ⁹individual described stem cell or 1 × 10 ¹⁰individual described stem cell.In other embodiment of the cell mass of any above-mentioned freezen protective, described stem cell by the number of times that goes down to posterity at least or be no more than 5 times, be no more than 10 times, be no more than 15 times, be no more than 20 times.In another embodiment of the cell mass of any above-mentioned freezen protective, described stem cell is increased in a reservoir.

4. accompanying drawing summary

Fig. 1 (A-B). (A) hESC through nurse cell and front-T-MSC stage differentiated be the schema of T-MSCs.The key organism mark that each differential period is correlated with as shown in the figure.(B) seed output and quality of the MSC that different MS C preparation method obtains is contrasted: three kinds of different methods induction hESCs differentiation.1) T-MSC: after trophoderm division culture medium cultivates 3 days, then cultivate 8-10 days at MSC growth medium.2) SB-MSC: after the division culture medium adding SB431542 cultivates 3-10 days, then cultivate 12 days at MSC growth medium.3) HB-MSC:hESC through the intermediate stage angioblast be divided into MSC, hESC serum free medium cultivate within 10-13 days, be divided into angioblast, then in MSC growth medium cultivate 12 days.In different culture media, total MSCs quantity (1,000,000 cells) within 10th day, 20 days and 30 days, is obtained after differentiation from differentiation method as shown in FIG..The cells ratio of the flow cytometry CD73 positive is to detect the purity of MSC.

Fig. 2 (A-C). in the cellular form change that hESCs observes to different time points in T-MSCs atomization.(A) second day: nurse cell; (B) the 5th day: pre-MSCs (mesoblastema); (C) the 9th day: MSCs.

Fig. 3 (A-C). express the ratio of the cell of nurse cell mark Trop-2 (Trp-2) and expression MSC mark CD73 to different time points analysis in T-MSCs atomization at hESCs.(A) the 2nd day: nurse cell; (B) the 5th day: pre-MSCs (mesoblastema); (C) the 9th day: MSCs.

Fig. 4 (A-H). at the expression pattern of differentiation T-MSC surface markers after 11 days.(A) Trp2 is the mark of nurse cell; (B) CD31 is the mark of endotheliocyte; (C) CD34 is the mark of hemopoietic stem cell; (D-H) CD73, CD90, CD 105, CD44 and CD29 are the marks of MSC.

The ion vitro immunization inhibit feature of Fig. 5 (A-R) .T-MSCs.BM-MSCs (G-L) or T-MSCs (M-R) mixes with the mouse lymphocyte of the ratio of 10:1 with mark CFSE.With the antibody irritation cell that the antibody of the CD3 of concentration 0.3 or 1 μ g/ml and concentration are the CD28 of 1 μ g/ml.Breed through the extent of dilution indicator cells of flow cytometry by CFSE.(A-F) shown in figure be the T cell of not adding BM-MSC or T-MSC (mark is contrasted).

Fig. 6 .T-MSC reduces the disease score of EAE mouse model.MOG35-55 adds that adjuvant and Toxins, pertussis process C57BL/6 build EAE mouse model.The 6th day of EAE mouse model induction, T-MSC, BM-MSC and through the MSC (hES-MSC (SB)) that obtains of SB431542 method induction hESCs in intraperitoneal injection of mice body.Injection after 27 days record disease score (from without disease 0 to serious disease 4).

Fig. 7 (A-C). detect T-MSC versatility, be divided into: (A) scleroblast, (B) chondrocyte, (C) adipocyte.

Fig. 8 .hES-HB-MSC (hES-hemagioblast source MSC)), T-MSC (MSC in hES-trophoblast source) and BM-MSC (MSC that adult bone marrow is originated) gene expression pattern compare of analysis.Genetic expression stdn, and show with any ceneme.

5. describe in detail

5.1 definition

Within the scope of the specific context of context of the present invention and each term application, the term applied in this specification sheets generally has their conventional sense in the art.Hereafter or other parts of the present invention carried out discussing describe method of the present invention and how to use them, for implementer provides extra guidance to some term.In addition, can be understood as, same thing can describe in more than one mode.Therefore, all can use optionally language and synonym to any one or the multiple term discussed herein, no matter whether this term describes in detail or is discussed at this, to it all without any special meaning.There is provided herein the synonym of some term.Describe one or more synonym and do not get rid of the synonym using other.The purposes of these or other embodiment Anywhere is in this manual only illustrative, and never limits scope and the implication of the present invention or any illustrational term.

Term hESC is human embryo stem cell, comprises the stem cell of the versatility produced from embryo, inner cell mass, blastomere or clone.

Term as used herein " hES-MSC " or " hES-MSCs " or " Human embryo mescenchymal stem cell " or " mescenchymal stem cell of derived from human embryonic stem " or " hES-MSC group " refer to mesenchymal stem cells, mesenchyme sample stroma cell, mescenchymal stem cell or mesenchyma stromal cells that human embryo stem cell or induced multi-potent stem cells " iPSCs " use any method derivative.HES-MSC used herein comprises that hES-MSC is unicellular, hES-MSC clone, a collection of hES-MSC, a large amount of hES-MSC or hES-MSC group.

Term " T-MSC " refer to human embryo stem cell ((hESC)) or induced multi-potent stem cells (iPSC) by the intermediate stage (the cell expressing Trop-2 in this stage, and there is nurse cell form) derivative MSC or mescenchymal stem cell/mesenchyma stromal cells.Term " hES-T-MSC " refers to the T-MSC that hESC breaks up." " iPS-T-MSC " or " iT-MSC " refers to the T-MSC that iPSC breaks up to term.Terminology used here " T-MSC " does not refer to nurse cell.If cell maintains at least one attribute in stem cell, such as, can be divided into the cell of other type of at least one, or similar function, so this cell is considered to " stem cell ".The description of these cells based on various structures and functional performance, can be included but not limited to, express or lack and express one or more marks.T-MSCs, comprises hES-T-MSC and iT-MSC, is versatility and can breaks up the cell or clone that produce other type.

The human pluripotent stem cells that term " hES-HB-MSC " or " HB-MSC " refer to comprise hESC and iPSCs is derivative mescenchymal stem cell by angioblast or blood vessel Colony forming intermediate steps.

Term used herein " clinical rank T-MSC " refers to the MSC with following characteristics: be adapted at clinically for people, birds or other animal.

Term used herein " T-MSC group " refers to a group T-MSC cell, comprises the T-MSC cell being suitable for treating and the T-MSC cell not being suitable for treatment.

Term used herein " clone that T-MSC is derivative " or " T-MSC-DL " refer to the cell that T-MSC breaks up or clone, include but not limited to adipocyte, sarcoplast, neural lineage cell, scleroblast, inoblast, chondrocyte and stroma cell.

Phrase used herein " treatment significant quantity " refers to the clinical remarkable symptom of the improvement experimenter that its quantity is enough to cause, or postpone or minimize or alleviate one or more symptoms relevant with described disease, or the beneficially altering causing experimenter desired in a physiologically.

Term " treatment " refers to the symptom slowing down, alleviate, improve or alleviate at least one disease, or after morbidity, reverse its outbreak.

Term " prevention " refers to work at premorbid, to prevent from forming this disease or the severity palliated a disease or delaying its process developed.

The term " experimenter " used in the application refers to have immune animal, such as bird and Mammals.Mammals comprises Canis animals, feline, rodent, ox, horse, pig, sheep and primate.Bird includes but not limited to poultry, song bird and the bird of prey.Therefore, the present invention can be used in veterinary medical, such as, in order to treat pet animal, farm-animals, the laboratory animal in animal park and animal in the wild.The present invention is particularly suitable for human medical's application.

Term " needs it " and is experimenter known or suspect suffer from or riskyly develop into disease, include but not limited to the autoimmune disease that multiple sclerosis is relevant with other T cell or the disease relevant to central nervous system, blood brain barrier or blood-spinal cord barrier.

One needs the curee for the treatment of to be the experimenter that disease has occurred.One needs the experimenter of prevention to be the experimenter with the risk factors that this disease occurs.

Term used herein " medicament " refers to the material that a kind of generation maybe can tell on, and can include but not limited to, chemical, pharmaceuticals, medicine, biotechnological formulation, small molecules, antibody, nucleic acid, peptide class and protein.

As used herein, stem cell is specific markers " positive ", refers to that this mark is when can detect.Such as, T-MSC is, the such as CD73 positive, because the amount of CD73 is detectable in T-MSC, higher than background (such as, isotype controls).Cell also can be that mark is positive, and when described mark can be used for the cell distinguishing at least one other types, or can be used for selecting or being separated this cell, wherein said mark exists or is expressed in this cell.

As used herein, " immunomodulatory " refers to and causes or have the ability to cause the detectable change of immune response, and cause immunne response can the ability of change detected.

As used herein, " immunosuppression " refers to and causes or have the ability to cause the detectable decline of immune response, and causes the ability of detected suppression of immunne response.

The present invention is trophocyte's differentiation that can derive from hESC based on Late Cambrian mescenchymal stem cell (MSCs), and the MSCs of this trophoblastic origin (T-MSC) can be used for tissue repair and immunoregulation effect.These T-MSC produced by disclosure method are the propagation of suppressor T cell and differentiation significantly in vitro, and reduce disease score in vivo, but the mescenchymal stem cell of derived from bone marrow (BM-MSC) does not have effect in vivo completely, although BM-MSC partly can lower T cell propagation and differentiation in vitro.T-MSC and BM-MSC as herein described compares, and has wonderful higher immunosuppressive activity.Method disclosed herein is efficient, and can with a large amount of highly purified T-MSC of the low production of cost.Method disclosed herein is highly repeatably, has very little difference between batches, and is easy to adapt to clinical needs.

Therefore, the present invention, by a kind of method providing human embryo stem cell to produce mescenchymal stem cell (MSC) in vitro, overcomes the problems referred to above.Allowed the production of these cells by the ability disclosing method generation hES-T-MSC herein, these cells can be used for various treatment use, comprise treatment and the prevention of multiple sclerosis and other autoimmune disorder.In addition, the hES-MSC produced by methods described herein has the ability through blood brain barrier (BBB) and blood-spinal cord barrier, and then allows them for various treatment use, comprises drug delivery.Method of the present invention provides further effectiveness, because they can produce spendable hES-T-MSC in business scope in a large number.

5.2 embryonic stem cells obtain T-MSC through nurse cell differentiation

Open a kind of nurse cell raw from embryonic stem cell (hES) Xing produces and obtains and the method for the mesenchymal stem cells that increases (MSCs) herein.These cells produced are defined as T-MSC.Separable and/or these T-MSC of purifying.

Embryonic stem cell has been in the news and has obtained cell (the Barbieriet al. (2005) of mescenchymal stem cell sample through diverse ways induction; Olivier et al. (2006); Sanchez et al. (2011); Brown et al. (2009)).But all these methods relate to Dual culture and manual selecting step, the output of restrictive cell and purity, the quality of gained cell is thus caused to differ.

Although hESC expresses low-level major histocompatibility complex antigen, have been found that the various kinds of cell expression level that these antigens break up at hESC higher (Draper et al., 2002; Drukker et al., 2006; Drukkeret al., 2002), therefore, these Transplanted cellss broken up are caused to patient body immunological rejection are paid close attention to greatly.On the contrary, MSC expresses low-level costimulatory molecules and major histocompatibility complex antigen, and has been applied to of the same race or heteroplastic transplantation model to treat autoimmune disorder (Gordon etc., 2008b; Grinnemo etc., 2004; Rafei etc., 2009a; Rafei etc., 2009b; Tse etc., 2003).T-MSC is the same with the MSC that adult tissue originates, and expresses low-level costimulatory molecules and major histocompatibility complex antigen, and does not need long-term engraftment just can play immunosuppressive action.Because the major histocompatibility complex antigen of MSC and patient does not mate, therefore without the need to worrying immunological rejection.HESC system is just enough to the large-scale T-MSC producing unlimited supply, and easy Mass Control, is applicable to suitability for industrialized production, becomes a kind of potential therapy of patient for the treatment of MS and other autoimmune disorderss based on T cell.

People's nurse cell can produce from human embryo stem cell.These embryonic stem cells comprise source or use, the ICMs of such as blastocyst, inoculation, one or more blastomere, or implant embryo or embryo's sample tissue of last stage, no matter whether produced by fertilization, body-cell neucleus transplanting (SCNT), parthenogenesis, the vegetative method of androgenesis.

Additionally or alternatively, nurse cell can produce from other embryonic derived cell.Such as, no matter whether nurse cell (but not necessarily needing by this step of embryonic stem cell), by the embryo originated or use is inoculated, ICMs, blastocyst, one or more blastomere, cytotrophoblast stem cells, embryonic genital cell or embryo or the embryo's sample tissue of implanting the last stage, pass through fertilization, body-cell neucleus transplanting (SCNT), parthenogenesis, the vegetative method generation of androgenesis.Similarly, the part cell from embryo-derived cells differentiation or clone generation nurse cell can be used.Such as, if a human embryonic stem cell is used to produce grow more original cell than nurse cell, so with regard to developmental potentiality and plasticity-, the cell of this embryonic origin can produce nurse cell.

Additionally or alternatively, nurse cell can, from the front raw material with enclosing postpartum of other childbirth, include but not limited to produce in umbilical cord, Cord blood, amniotic fluid, amnion stem cell and placenta.

Embryonic stem cell can as the parent material of this method.Embryonic stem cell by the current known any one cultural method in this area, such as, can be with or without feeder cell.

In embodiment set forth herein, employ 8 kinds of hESC clones altogether, be respectively H9 (WiCell ResearchInstitute) (Thomson etc. (1998)), CT2 (University of Connecticut Stem Cell Core (Lin etc. (2010)), ES03-Envy (Envy, with the clone of GFP label, ES International) and (Costa etc. (2005)), ESI-017, ESI-053, ESI-049, ESI-035 and ESI-051.

Present method obtains in first step of T-MSC, human embryo stem cell is cultivated at the serum free medium not containing bFGF with little agglomerate or unicellular form, then cell is spread again and cultivate to containing continuing in the substratum of BMP4 (1-200ng/ml), select BMP4 just (2-5 days) highly purified, to express typical trophoblast marker Trop2/TACSTD2 (Trp2) nurse cell can be obtained at short notice because only add this cytokine.Add the production rate that TGF beta inhibitor (SB431542 (1-20 μM), A83-01 (0.2-5 μM) or ALK5 inhibitor (1-20 μM), etc.) can improve nurse cell.Cell increased and is divided into the nurse cell with trophoderm form in 2-5 days.In some embodiments, the cell expressing Trop-2/TACSTD2 (Trp-2) (Xu etc., 2002) more than 90%.Nurse cell can be separated according to cell size or adopt antibody purification, as immune affinity column chromatography.

In one embodiment, nurse cell is digested to unicellular with pancreatin surrogate TrypLE, pancreatin or Gelatinase B.Resuspended in the unicellular mescenchymal stem cell substratum optimizing obtained, as contained 2-20% foetal calf serum (FBS) or the α-MEM substratum of people AB serum (ABHS), the DMEM in high glucose substratum containing 2-20%FBS or ABHS.The available 5-20% serum substitute (KOSR) or bovine serum albumin (BSA) or other available commercialization serum-free MSC substratum any of containing substitutes FBS.In some embodiments, in substratum, the concentration of serum, KOSR or BSA is about 5-20%.In some embodiments, FBS is preferred.In some embodiments, the culture density of cell is about 10-1000 cell/cm ².In some embodiments, cell cultures at the extracellular matrix environment of simulated tissue, as gelatin, vitronectin, ln, fibronectin and collagen I.In some embodiments, the substratum of MSC adds LIF (2-20ng/ml), bFGF (2-100ng/ml) or PDGF (1-50ng/ml) to improve amplification efficiency.

After resuspended about 24 hours, many cells (50-90%) are adherent in culture dish.After about 2-3 days, nurse cell starts-T-MSC differentiation forward, and these cellular fories are elongated and edge is transparent.In some embodiments, front-T-MSC expresses CD73 and Trop-2 simultaneously.After 6-10 days, be divided into the mesenchymal stem cells of spindle sample form more than the nurse cell of 80-90%, namely so-called T-MSC.T-MSC, by detecting the expression identification of some mark, as expressed CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and CD166, and does not express or expresses low-level CD31, CD34 and CD45.In some embodiments, T-MSC does not express HOX and HLA-G.In some embodiments, T-MSC expresses high-caliber CXCR7, CXCL2 and CXCL12, but expresses other gene of low-level HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and HOX family.T-MSC also has following characteristics simultaneously: versatility also can be divided into adipocyte, chondrocyte, scleroblast, neurone, sarcoplast, stroma cell and inoblast.

There is provided a kind of cell mass of separation herein, wherein, described cell mass comprises multiple T-MSC with immune suppression function.These T-MSC at least express a kind of following mark: CD73, CD90 and CD105.

In the further embodiment of the present invention, extra step of adding radiation T-MSCs.This irradiation can adopt any method of radiating known in the art, includes but not limited to gamma radiation, as caesium-137 gamma radiation, or the radiation of X-ray photons.The preferred amounts of radiation is between 5 and 20000gy, preferred between 50 and 100gy, most preferably 80gy.

In one embodiment, method as herein described is the method for a kind of new hESCs derivative (herein also referred to as producing) T-MSC.The method comprises the following steps:

A. cultivate in serum free medium and comprise the cell culture of human embryo stem cell, in substratum, add at least one somatomedin, and content is enough to inducing embryo stem cell is divided into nurse cell.In one embodiment, the time cycle being divided into nurse cell is about 2-5 days.In one embodiment, substratum is containing BMP4, interpolation or do not add TGFb inhibitor (such as SB431542, A83-01 or ALK5 inhibitor, etc.) and can improve differentiation efficiency.

B. at least add a kind of long-living factor to the cell culture containing nurse cell also to continue to cultivate in serum free medium, the amount of adding somatomedin here enough promotes that nurse cell increases.In one embodiment, substratum contains BMP4 (this step is optional).

C. the nurse cell be separated spreads to bag again by the culture dish of gelatin, ln, fibronectin, vitronectin, collagen or matrigel, and cultivation is containing or is not containing in the substratum of serum, wherein, these substratum with amount be enough to promote nurse cell be divided into T-MSC by the front-T-MSC intermediate stage.In one embodiment, the nurse cell of separation produces the T-MSC expressing adult MSCs mark after cultivating 6-10 days.Wherein express the cell surface marker of adult MSCs at least about the T-MSC of 90%, 95%, 96%, 97%, 98%, 99%.In one embodiment, LIF, bFGF, PDGF is added in substratum to promote amplification efficiency.Wherein, the T-MSC of acquisition expresses adult MSCs cell surface marker at least about 90%, 95%, 96%, 97%, 98%, 99%.

As depicted in figs. 1 and 2, first hESC breaks up as little agglomerate or unicellular by disclosure method, is then again spread by these cells and cultivates at the substratum only containing a kind of cytokine BMP4 and a kind of TGF beta inhibitor.The TGF beta inhibitor added (2-5 days) can obtain the nurse cell group of height homogeneity within the time, and these cell masses express typical nurse cell mark Trop-2/TACSTD2 (Trp-2) (Xu etc., 2002).Then nurse cell is broken up, again bag is layered on by the culture dish of gelatin, ln, fibronectin, vitronectin, collagen or matrigel, and cultivate in MSC growth medium and produce fusoid cell in 4-10 days, form and the typical MSCs of these cells are similar.

Method disclosed herein, is different from other method, does not need feeder layer cells, does not need sorting or select cell by hand.The first step of nurse cell differentiation is carried out in definite ingredients, the serum free medium not containing bFGF.Whole differentiation flow process only needs 2 steps, total 6-14 days consuming time, just can produce high yield and highly purified T-MSC (Fig. 1).This is that the hESC of at present report is divided into shortest time in MSC method.With compared with the method for living, output and the purity of the T-MSC of the method acquisition are all very high.In 30 days, the T-MSC quantity of acquisition is 5 × 10 of initial hESCs ⁵doubly, and the ratio of CD73 positive cell is very high, and wherein CD73 is the specific mark of MSC.But the quantity of the MSC that other method obtains, compared with the quantity of initial hESCs, be less than 100 times, and the ratio of the CD73 positive is low in the cell obtained.Induce containing the two cell that is positive, the intermediate stage of CD73/Trp-2 in the T-MSC that obtains, hereinafter referred to as front-T-MSC.Through the process of BMP4 and TGF beta inhibitor after 2-3 days, first these cells express Trp-2, and wherein the cells ratio of the Trp-2 positive is very high, and show nurse cell form homogeneity (Fig. 2 and 3).After 5-6 days, cell expresses Trp-2 and CD73 simultaneously, after 6-14, cell no longer expresses Trp2, but express the typical cell surface marker CD73 of MSC, CD90, CD105, CD44 and CD29, and ratio is very high: CD73 (>98%), CD90 (>95%), CD105 (>90%), CD44 (>95%), CD29 (>80%), these cells are CD31 simultaneously, CD34 and CD45 is negative, wherein CD31 is endothelial cell marker, CD34 and CD45 is hemopoietic stem cell marker (Fig. 3 and 4).

Utilize the T-MSC produced in method of the present disclosure can be divided into the scleroblast system in downstream, chondrocyte system and adipose cell lines.Therefore, these T-MSC are similar to marrow and other tissue-derived MSC s from morphology and function.

The mescenchymal stem cell in 5.3 human embryo stem cell sources

The mescenchymal stem cell (BM-MSCs) of derived from bone marrow is used to animal model and the clinical trial for the treatment of many autoimmune disorders for a long time.But in some reports, the immunosuppressive action of BM-MSCs is also inconsistent, shows that BM-MSCs can not treat some autoimmune disorder (Tyndall, 2011) effectively.The data of contrast BM-MSCs and T-MSC suppressor T cell propagation and follow-up φt cell receptor activation capability are provided herein.As shown in Figure 7, when anti-CD 3 antibodies processes with lower concentration (0.3ug/ml), BM-MSCs can suppress the propagation of CD4 and CD8 positive cell simultaneously, similar with the result of T-MSC.But when the concentration of anti-CD 3 antibodies brings up to 1ug/ml, compared with T-MSC, BM-MSCs suppresses the ability of the propagation of CD4 and CD8 positive cell lower.Here adopt CFSE dilution experiment to detect the propagation of T cell: increase the weakening along with CFSE signal of T cell ratio, show that propagation is accelerated.As shown in Figure 5, when the concentration of anti-CD 3 antibodies brings up to 1ug/ml, detect and find that the signal of CFSE in the T cell of the 59%CD4 positive and the T cell of the 46%CD8 positive declines.In T-MSC group, the ratio of the T cell of CD4 and the CD8 positive is all reduced to 16%; In BM-MSC group, the ratio of the T cell of CD4 and the CD8 positive is reduced to 32% and 36% respectively.

The immunosuppressive activity external with T-MSC is consistent, and the T-MSC that produces of disclosure method can Experiment on therapy Autoimmune Encephalomyelitis (EAE) effectively, and this is a kind of mouse model of multiple sclerosis.As shown in Figure 6, compared with control group, decline significantly at the disease score of EAE mouse model induction latter 6 days injection T-MSC, EAE mouse.

The feature that the cell of disclosure method production is other is: T-MSC has stronger immunosuppressive activity (Chen et al., 2012) (Fig. 6) than BM-MSCs with from the hES-MSCs of SB431542 process.Repeat in experiment several, BM-MSCs can not lower the disease score of EAE mouse all the time.Therefore, the T-MSC applying the production of disclosure method clinically substitutes BM-MSCs, can eliminate the risk that invasive bone marrow aspiration brings, and reduces the time waiting for marrow donations, reduces cost, is reduced to the batch wise differences of the BM-MSCs that different patient prepares.

In a word, there is provided herein a kind of method that hESCs of utilization efficiently produces the cell of interstitial like cell or MSCs, and use these MSCs to treat autoimmune disorder, the feature of the method is, through this intermediate stage of nurse cell in hESCs atomization.Microarray results shows, although T-MSC and BM-MSCs can be divided into same downstream cellular system (Fig. 7), and the gene expression pattern of T-MSC and BM-MSCs's and inconsistent (data do not provide).In addition, in vivo with external, stronger than BM-MSCs of the immunosuppressive activity of T-MSC.

Available data shows, the T-MSC that disclosure method is produced is different from traditional adult MSCs.Because T-MSC has the ability of powerful suppressor T cell propagation, T-MSC may have better effect than use BM-MSCs when treating multiple sclerosis.In order to solve potential safety problem, T-MSC being injected to the SCID beige mice of immune deficiency, in mouse, not observing teratoma formed.

The T-MSC that the present invention produces is unique, and has multiple therepic use and other purposes.Therefore, the present invention includes several formulations, comprise pharmaceutical preparation and the composition comprising T-MSC.

Term " trophoderm-mescenchymal stem cell (T-MSC) " refers to that described nurse cell is expressed Trop-2 and had nurse cell sample form from human embryo stem cell (hESC) or induced multi-potent stem cells (iPSC) through the derivative mescenchymal stem cell of nurse cell intermediate product or mescenchymal stem cell/stroma cell.Term " human embryo stem cell-trophoderm-mescenchymal stem cell (hES-T-MSC) " refers to the T-MSC deriving from hESC.Term " iPS-T-MSC " and " iT-MSC " refer to the mescenchymal stem cell from induced multi-potent stem cells differentiation.Term " T-MSC " does not refer to nurse cell here.If cell has the attribute of a kind of stem cell at least, so this cell is considered to stem cell.Described attribute as, have the ability to be divided into the cell of at least one or other type, like this.To the description of these cells based on numerous structure and function characteristic, include but not limited to: express or do not express one or more marks.Particularly, T-MSC has the characteristic of the minicell body of inoblast form.T-MSCs comprises hES-T-MSC and iT-MSC, has versatility and can break up the cell and clone that produce other type.Term " T-MSC-DL " refers to all cells that T-MSC breaks up and clone.

Differentiation method as herein described can obtain the MSC from iPS cytodifferentiation in 6-14 days, and this is the shortest time of report at present.Therefore, these iT-MSC can be used for the specific iPS therapy of patient, particularly need to produce MSC in emergency circumstances within very short time, the treatment etc. of such as acute cardiac infraction, acute heart failure, acute spinal cord injury and acute radiation/burning.

Can by the expression of DNA, RNA or albumen that detects one or more marks or not expression identification T-MSC.The qualification of T-MSC is by detecting cell surface marker CD73 or expressing at least one or multiple following cell surface marker: CD90, CD105, CD13, CD29, CD54, CD44, CD146 and CD166, or does not express or the following at least one cell surface marker of low expression level: CD34, CD31 and CD45.

Selectively or extraly, the qualification of T-MSC can express low-level one or more proinflammatory proteins following according to them: MMP2, RAGE, IFNGR2, TNF α, IL-12A, IL-6 and CAM1.Above-described gene expression pattern is compared with mesenchymal stem cells MSCs.Especially, IL-6 at the expression amount of BM-MSCs higher than T-MSC.IL-6 is a kind of cytokine of multiple-effect, participates in hematopoietic cell, cross action between immunocyte and stroma cell, comprises generation and the termination of inflammation.

The characteristic of described T-MSC is also: their capable propagation suppressing the T cell after being upset in vitro.This characteristic and BM-MSCs can not suppress the propagation of the T cell after being upset to be formed to contrast in vitro.

Therefore, T-MSC as described herein has following a kind of characteristic at least: (1) is divided into adipocyte, chondrocyte, neurone, sarcoplast, stroma cell and inoblast; (2) there is the form of fibroblast-like cells; (3) do not express or express low-level CD34, CD31 and/or CD45; (5) do not express or express low-level MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and/or VCAM1, particularly IL-6; (6) low-level MHC antigen HLA-G and/or HLA-ABC is expressed, or not expression of HLA-DR and/or CD80; (7): the propagation suppressing the T cell after being upset in vitro.In some embodiments, described T-MSCs has at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds or all 7 kinds of characteristics.

In some embodiments, T-MSC is different from the HB-MSC of previously report.The expression level of CXCR7, CXCL2 and/or CXCL12 of T-MSC at least doubles than HB-MSC.But at least few than the T-MSC half of the expression level of other genes of HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and HOX family of T-MSC.

In addition, that T-MSC has is unique, through the ability of hemato encephalic barrier and blood spinal cord barrier, this penetrativity makes this cell be particularly suitable for treatment and diagnostic use.T-MSC of the present invention has migration the turnover blood vessel of spinal cord, the ability through blood spinal cord barrier, and to play a role in central nervous system, this effect includes but not limited to send the medicine being used for the treatment of and diagnosing.This and BM-MSCs do not have this function and present a contrast.

Another embodiment of the invention is radiation T-MSC.The feature of this embodiment is, is had above at least a kind of cited characteristic, at least 2 kinds of characteristics, at least 3 kinds of characteristics, at least 4 kinds of characteristics, at least 5 kinds of characteristics, at least 6 kinds of characteristics or at least all 7 kinds of characteristics by the T-MSC of radiation.

In another embodiment, described cell culture comprises T-MSC.In some embodiments, described T-MSC is divided into adipocyte, chondrocyte, scleroblast, neurone, sarcoplast, stroma cell and inoblast.In some embodiments, described T-MSC expresses CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and/or CD166.In some embodiments, described cell expressing low-level or do not express CD34, CD31 and/or CD45.In some embodiments, described cell expressing low-level or do not express MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and/or VCAM1, particularly IL-6.At some in other embodiment, described cell expressing major histocompatibility complex antigen HLA-G and/or HLA-ABC, and express low-level or non-expression of HLA-DR and/or CD80.In some embodiments, described cell suppresses the propagation of the T cell after being upset in vitro.In some embodiments, described cell can pass hemato encephalic barrier and blood spinal cord barrier.In some embodiments, described cell is by radiation.

On the other hand, a kind of pharmaceutical preparation comprising T-MSC. is disclosed herein.In some embodiments, described T-MSC is divided into adipocyte, chondrocyte, scleroblast, neurone, sarcoplast, stroma cell and inoblast.In some embodiments, described T-MSC expresses CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and/or CD166.In some embodiments, these cell expressing low-levels or do not express CD34, CD31 and/or CD45.In some embodiments, described cell expressing low-level or do not express MMP2, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α, IL-6 and/or VCAM1, particularly IL-6.In some embodiments, described cell expressing major histocompatibility complex antigen HLA-G and/or HLA-ABC, and express low-level or non-expression of HLA-DR and/or CD80.In some embodiments, described cell can pass through hemato encephalic barrier and blood spinal cord barrier.In some embodiments, described cell is by radiation.Any pharmaceutically acceptable carrier and adjuvant can be used to prepare described pharmaceutical preparation.

There is provided multiple T-MSC herein, wherein, these T-MSC directly obtain from human embryonic stem cell and are separated.These human embryonic stem cells have been gone down to posterity 1,2,3,4,5,6,7,8,9,10,12,14,16,18,20,25,30 or more algebraically or its combination.

In some embodiments, the cell of a kind of freezing T-MSC or part or end differentiation is eventually provided herein.

In some embodiments, provide the medical use of T-MSC herein, or T-MSC composition, or T-MSC preparation, comprise by the T-MSC of radiation.These cells and medicament may be used for the disease described in treatment, and also can be applied to delivery system, these medicines need to pass through hemato encephalic barrier and blood spinal cord barrier.

In some embodiments, the invention provides the T-MSC of the nurse cell of the freezen protective of preparation, front-T-MSC, part or wherein whole terminal differentiation.

In some embodiments, the invention provides the medical use of T-MSC, or composition, or T-MSC preparation, comprise by the T-MSC of radiation.These cells and medicament may be used for treating the disease described in detail in any described symptom or whole specification sheets, and also can be applied to delivery system, these medicines need through hemato encephalic barrier and blood spinal cord barrier.

5.4 select and produce T-MSC group

Here a kind of method of T-MSC providing discriminating to have hyperimmunization to suppress effect, wherein, by differentiating the expression pattern of the biomarker of T-MSC, described T-MSC be have hyperimmunization suppress effect, clinical grade other and be applied to treatment.In some embodiments, other T-MSC of these clinical grade has following characteristic: (i) comprises cell expressing group-1 mark of >95%; (ii) cell expressing group-2 mark of >80% is comprised; (iii) cell expressing group-3 mark of <5% is comprised; (iv) IL-10 and TGF β is expressed; V () comprises cell expressing IL-6, IL-12 and TNF α of <2%; (vi) mark of the cell coexpression all groups-4 of <0.001% is comprised.Here group-1 mark is: CD73, CD90, CD105, CD146, CD166 and CD44.Group-2 mark is: CD13, CD29, CD54 and CD49E.Group-3 mark is: CD45, CD34, CD31 and SSEA4.Group-4 mark is: OCT4, NANOG, TRA-1-60 and SSEA4.

In some embodiments, described method comprises and detects encoding anti-inflammatory inflammation factor (AIF) and the differential expression that marks of proinflammatory inflammation factor (PIF).In some embodiments, described anti-inflammatory cytokine is IL-10 and TGF β 2.In some embodiments, the up-regulated of proinflammatory inflammation factor.In some embodiments, the expression amount being more than marked at T-MSC is at least 1.5 times of BM-MSC.In some embodiments, proinflammatory inflammation factor is IL-6, IL-12, TNF α, CCL2, VCAM1, RAGE and MMP2.In some embodiments, the expression of proinflammatory inflammation factor declines.In some embodiments, the expression amount of T-MSC is more than marked at least than few half of BM-MSC.In another embodiment, the cells ratio with the IL-6 positive of the T-MSC of hyperimmunization rejection ability is less than 5%, 4%, 3%, 2% or 1%.In some embodiments, T-MSC may express high-caliber TGF β 2 and IL-10.In some embodiments, the expression of described mark take BM-MSC as contrast.

The qualification program of other T-MSC group of clinical grade is provided here.In order to determine whether T-MSC group is applicable to application for the treatment of, need the expression detecting cell mass specific biomarker.These biomarkers comprise such as, the mark of (1) mescenchymal stem cell: CD73, CD90, CD105, CD166 and CD44 (first group); (2) mark of mescenchymal stem cell: CD13, CD29, CD54, CD49E, SCA-1 and STRO-1 (second group); (3) mark of hematopoietic stem/progenitor: CD45, CD34, and the mark CD31 of endotheliocyte; (4) immune labeled: HLA-ABC, HLA-G, CD80 and CD86; (5) cytokine: IL-10, TGF β, IL-6 and IL-12; (6) mark of versatility: OCT4, NANOG, TRA-1-60 and SSEA-4.In some embodiments, T-MSC group comprise more than 95%, 96%, 97%, at least one group-1 of cell expressing of 98% or 99% mark.In some embodiments, T-MSC group comprise more than 80%, 85%, 90%, at least one group-2 of cell expressing of 95% or 99% mark.In some embodiments, in some embodiments, T-MSC group comprises at least one group-3 of the cell expressing mark being less than 0.1%, 0.08%, 0.05%, 0.03%, 0.02% or 0.01%.In some embodiments, T-MSC group comprise more than 80%, 85%, 90%, the cell expressing IL-10 of 95% or 99% and/or TGF β.In some embodiments, T-MSC group comprises cell mass expression IL-6 and/or IL-12 being less than 5%, 4%, 3%, 2%, 1%.In some embodiments, T-MSC group comprises at least one the 6th group echo of cell expressing being less than 0.001%.In some embodiments, other T-MSC of clinical prime as positive control, compared with other T-MSC of clinical grade.In some embodiments, by the feature of polychrome flow cytometry and/or immunofluorescence analysis T-MSC.In some embodiments, T-MSC group expresses CCL2, CCL3, CCL4, CCL5, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF α, TGF β, IFN γ, GM-CSF, G-CSF, bFGF, CXCL5, VEGF, TPO or its combination.In some embodiments, also following analysis is done to T-MSC group: (1) exists foreign material as intracellular toxin and residual cytokine/somatomedin, and/or (2) gene unconventionality (utilizing karyotyping and genome sequencing).

Here provide another to the qualification program of clinical rank T-MSC group.The T-MSC having better regeneration potential and immune suppression function may express lower level CD29.Here, 1-2 is for the expression amount of the CD29 of T-MSC as background level, if in certain follow-up generation and program, the expression level of T-MSC raises 2 times, and so these cells will be stopped and go down to posterity.

Known in the art to the detection method of the gene expression pattern of T-MSC.These methods include but not limited to: flow cytometry, multiple chip, RT-PCR, RNA trace and western blotting.In some embodiments, the luminex system adopt the flow microsphere analyzed based on cytokine profiles to detect to the analysis of the gene expression pattern of MSC, analyzing based on cytokine profiles, the high throughput analysis of genetic expression, quantitative RT-PCR, ELISpot assay, cytokine enzyme-linked immunosorbent assay, luciferase assay, flow cytometry, fluorescence report system, tissue staining and immunofluorescence dyeing.

5.4.1 the method for nucleic acid biomarker is detected

In a particular embodiment, nucleic acid belongs to a kind of biomarker in biomarker spectrum.These biomarkers and biomarker are composed corresponding feature and can be produced by the following stated, as detected the gene expression product (as polynucleotide and polypeptide) of one or more biomarker.In a concrete embodiment; the acquisition channel that these biomarkers and biomarker compose corresponding feature is: utilize any method known in the art to detect and/or analyze and disclosed hereinly a kind ofly mark one or more expressed nucleic acid, and described method includes but not limited to hybridization, chip analysis, RT-PCR, nuclease protection mensuration and rna blot analysis.

In some embodiments, method and the composition of the present invention of detection and/or analysis of nucleic acids comprise RNA molecule, the RNA molecule (comprising mRNA molecule) such as expressed, the variable sheer of mRNA, rna regulation, cRNA molecule (as RNA molecule is transcribed into cDNA molecule in vitro) and its identification fragment.

In a particular embodiment, the nucleic acid prepared in vitro from there is cell culture nucleic acid, from cell culture be separated or part be separated nucleic acid, described cell culture is known in the art.The above general description such as, Sambrook etc., 2001, molecular cloning, laboratory manual, the third edition, CSH Press (cold spring port, New York), this file full content is hereby incorporated by.

5.4.1.1 nucleic acid array

In some embodiments, nucleic acid array, by detecting any one or more biomarkers described here, obtains the feature of the biomarker of biomarker spectrum.In an embodiment of the invention, microarray is utilized to detect the eigenwert of the biomarker of biomarker spectrum as cDNA microarray.Exemplary method and the embodiment of cDNA microarray analysis are below described.

In some embodiments, by obtaining the eigenwert of the biomarker that biomarker is composed, the nucleotide sequence of the mRNA transcript existed in the nucleic acid representative biological specimen of described mark with the nucleic acid hybridization of detectable mark on array, or consistent with these sequences.Described biological sample (if synthesis is from the fluorescently-labeled cDNA of sample) is hybridized mutually with the microarray comprising one or more probe spot.

There is diverse ways to prepare nucleic acid array as microarray, some of them method is described below herein.Preferably, array can copy again, allows the multiple copies producing given array also mutually to compare.Preferably, array is by material manufacture stable under (such as, the nucleic acid hybridization) condition of combination.Those skilled in the art will know to hybridize test probe to probe spot suitable support, matrix or carrier used on array, or routine experimentation can be utilized to determine these carriers be applicable to.

Array such as microarray can comprise one or more test probe in use.In some embodiments, each such test probe comprise one with the polynucleotide sequence of the subsequence complementation of RNA or DNA that will detect.Each probe has different nucleotide sequences usually, and the position of each probe on the solid surface of array normally known or confirmable.The array useful to the present invention can comprise, such as oligonucleotide microarray, array based on cDNA, single nucleotide polymorphism array, variable sheer volume array and any other qualitative, quantitative or semiquantitative array of the expression of mark as herein described can be provided.The microarray of some type is addressable array.More specifically, some microarraies are addressable arrays of location.In some embodiments, the position of each probe of array is known.Predetermined position on known solid carrier, thus the identity of each probe (such as, sequence) can from it on array the position of (such as, carrier or on the surface) determine.In some embodiments, these arrays are orderly arrays.Microarray general description in Draghici, 2003, DNA microarray data analysis tool, Chapman and Hall/CRC, this file full content is hereby incorporated by.

5.4.1.2RT-PCR

In some embodiments, for determining the eigenwert of the biomarker in the biomarker spectrum of the expression level of one or more mark described herein, this eigenwert is by using reverse transcription (RT), and combines the polymerase chain reaction (PCR) of cloning RNA from sample.According to this embodiment, reverse transcription can be quantitative or semiquantitative.Teaching herein, described RT-PCR method can use with the methods combining of above-mentioned microarray.Such as, the PCR reaction of a batch can be carried out, and PCR primer can be resolved and be used as probe spot on microarray.

Total serum IgE or mRNA is used to make template, and with the primer of mark (or multiple) transcribed portion specific pairs for starting reverse transcription.RNA reverse transcription is the method for cDNA is well-known, with reference to (calendar year 2001s) such as Sambrook above show.Design of primers can according to delivered or can from any sequence library that can openly obtain, as the known nucleotide sequence in GenBank realizes.The primer for any mark as herein described can be designed.In addition, the software design primer (such as, Primer Designer 1.0 and Scientific Software etc.) be purchased can be utilized.The product of reverse transcription is used as the template of PCR subsequently.

PCR provides a kind of by using the method for rapid amplifying specific nucleic acid sequence of the interested target sequence that to be increased by multiple circulations of DNA replication dna that are heat-staple, that rely on the archaeal dna polymerase catalysis of DNA.PCR reaction needed exists: the nucleic acid be amplified, two the single stranded oligonucleotide primers of nucleic acid flank be amplified, archaeal dna polymerase, deoxyribonucleoside triphosphate damping fluid and salt ion.The method of round pcr is technology known in the art.Can with reference to such as Mullis and Faloona to the use of PCR, 1987, Enzymology method, 155:335.This file full content is hereby incorporated by.

Pcr amplification needs template DNA or cDNA (at least 10fg, more effectively, 1-1000ng) and Oligonucleolide primers (at least 25pmol).Typical reaction mixture comprises: Oligonucleolide primers, the 2.5 μ l 10M PCR of 2 μ l DNA, 25pmol cushion 1 (Perkin-Elmer, Foster city, California), the dNTP of 0.4 μ l 1.25M, 0.15 μ l (or 2.5units) Taq archaeal dna polymerase (Perkin-Elmer, Foster city, California) and add deionized water to cumulative volume 25 μ l.Mineral oil covers PCR reaction system, is carrying out PCR reaction with programmable heat circulating instrument.

Quantitative RT-PCR (" QRT-PCR ") essence is quantitative, also can provide the quantitative measurment to marker expression level.The reverse transcription of QRT-PCR and PCR reaction can be carried out in two steps, or these two reactions can occur simultaneously.The wherein antisense probe of a technology use specific transcription, usually commercially available test kit is had, as Taqman test kit (the Perkin Elmer of AppliedBiosystems (Foster city, California), Foster city, California).This probe is specific PCR primer (such as, the nucleic acid fragment from gene), and containing quencher and complexing to the fluorescent reporter group of described oligonucleotide 5' end.Different fluorescent marks is connected on different probes, thus allows to measure two kinds of probes in a reaction.When Taq archaeal dna polymerase is activated, it utilizes its 5' to 3' 5 prime excision enzyme activity to remove the fluorescent reporter group be incorporated in template.When there is not described quenching group, reporter group is luminous.The change of probe color is directly proportional to the content of each specific product, and is measured by photofluorometer.Therefore, the amount of often kind of color can be measured and PCR primer is entered quantitatively raw.Described PCR reaction is carried out, to detect the sample of many different sourcess simultaneously in 96 orifice plates.Described Taqman system has the attendant advantages not needing gel electrophoresis, and can quantitative analysis when using typical curve.

The second technology, uses intercalative dye to detect PCR primer quantitatively.This dyestuff is as commercially available QuantiTectSYBR Green PCR (Qiagen company, Valencia, California).In PCR step of reaction, SYBRGreen dyestuff mixes in the PCR primer of double-strand, and the fluorescent signal produced is directly proportional to the amount of PCR primer.

Taqman and QuantiTect SYBR can be used after RNA reverse transcription.Reverse transcription both in the step (single stage method) of identical reaction system as PCR, also can complete (two-step approach) before pcr amplification.In addition, the system of other known detection by quantitative mrna expression comprises Molecular Beacons, and the probe that this system uses has fluorescence molecule and quencher molecule, and this probe can form hairpin structure, when hair clip is formed, and fluorescence molecule cancellation; When probe hybridization is to PCR primer, send fluorescent signal, according to the quantification of intensities ground gene expression detection of hybridization fluorescent signal.

5.4.1.3RNA blot analysis

Any known hybridization technique in this area can be used for the eigenwert of the biomarker producing biomarker spectrum.In other are specifically implemented, obtain eigenwert, rna blot analysis and the quantitatively special RNA molecule of the biomarker in biomarker spectrum by rna blot analysis.The RNA trace of a standard can be used for the size determining a kind of rna transcription thing, and the rna transcription thing of qualification alternative splicing, identifies one or more gene as herein described relative quantity in the sample to which (especially mRNA).According to the conventional RNA hybridization technique known to those of ordinary skill in the art, in RNA trace, RNA sample is electrophoresis on the sepharose of sex change first, according to size separation RNA, is then transferred on film by RNA, is cross-linked with the probe of mark and hybridizes.Heterotope or highly active radiolabeled probe can be used, comprise DNA probe and the rna probe of transcribing in vitro and oligonucleotide that random that cause, nick translation or PCR produces.In addition, only homeologous sequence (such as, may contain an exon from the cDNA of different species or genomic DNA fragment) can be used as probe.The probe of mark, such as radiolabeled cDNA, or comprise the DNA of total length, strand, or be the fragment of this DNA sequence dna, wherein length can be at least 20, at least 30, at least 50 or at least continuous 100 Nucleotide.By the many different methods label probes known by any those skilled in the art of the present technique.The marks being most commonly used to these researchs are radioelement, enzyme, can send when being exposed to ultraviolet the chemical substance of fluorescence and other.Many fluorescent substances are known and can with marking.These include but not limited to fluorescein, rhodamine, auramine, Texas is red, AMCA is blue and fluorescent yellow.Radio-labeling can by any at present can method of counting detect.Isotropic substance includes but not limited to ³h, ¹⁴c, ³²p, ³⁵s, ³⁶cl, ⁵¹cr, ⁵⁷co, ⁵⁸co, ⁵⁹fe, ⁹⁰y, ¹²⁵i, ¹³¹i and ¹⁸⁶re.Enzyme labelling is also available, and can be detected by any existing colorimetry, spectrophotometry, fluorimetry, amperometry or gasometric techniques.Enzyme by with bridging molecules as reactions such as carbodiimide, vulcabond, glutaraldehyde, be coupled to the particle of selection.Any one enzyme well known by persons skilled in the art can be used.The example of these enzymes includes but not limited to peroxidase, beta-D-galactosidase, urase, glucose oxidase, peroxidase and alkaline phosphatase.Can with reference to U.S. Patent number 3654090,3850752 and 4016043.

5.4.2 the method for albumen is detected

In the specific embodiment of the present invention, the biomarker characteristic value of biomarker spectrum can be obtained by detecting protein, such as, by detecting the protein of the expression product (as nucleic acid or protein) of one or more marks as herein described or the protein of posttranslational modification or other modification or the protein through processing.In a concrete embodiment, identification fragment by detecting and/or analyze one or more protein and/or marker expression disclosed herein produces biomarker spectrum, wherein, the method of detection albumen is any method known to those skilled in the art, includes but not limited to protein microarray analysis, immunohistochemistry and mass spectroscopy.

The standard technique of immunoassay can be used for detecting interested albumen in the amount of protein and cell culture.Such as, first carry out SDS-PAGE (SDS-PAGE), then carry out western blotting and co-immunoprecipitation and detect interested albumen in the amount of protein and sample, other detection method also has immunocytochemistry etc.A kind of exemplary agents for testing goal protein is the antibody that can be specifically bound to interested albumen.No matter the antibody of preferred detectable label is direct or indirect labelling.

For such detection method, technology known in the art is utilized to be easy to be separated the protein obtaining wanting to analyze in cell culture.Method for protein isolation can reference, such as Harlow and Lane, and 1988, antibody: laboratory manual, CSH Press (cold spring port, New York), this file full content was hereby incorporated by.

In some embodiments, the method detecting protein or target protein relates to the interaction of protein-specific antibody.Such as, antibody is for interested albumen.Standard technique well known in the art can be utilized to produce antibody.In a particular embodiment, antibody can be polyclonal, or more preferably monoclonal antibody.Complete antibody or antibody fragment (such as, single-chain antibody, Fab or F (ab') can be used ₂.

Such as, can the antibody of specific binding target protein or fragment can be used for quantitatively or the existence of qualitative detection protein.The above can be realized by such as immunofluorescence technique.In addition, antibody (or its fragment) can be used at histology situ testing goal albumen, as immune fluorescence or immunoelectronmicroscopy.By the biological specimen (as biopsy sample) got from patient, and in situ detection is completed to the antibody that it adds for proteins of interest.Administration of antibodies (or fragment) preferably makes antibody (or fragment) cover biological specimen.By using such method, in specific sample, likely not only detecting target protein whether exist, also detecting its distribution.Multiple known histological method (as dyeing process) can realize this in situ detection.

The immunoassay of testing goal albumen generally includes antibody and the sample incubation of detectable label, and this antibody can identify target protein.In addition, immunoassay also comprises the antibody utilizing any technology for detection known in the art to combine.For discussing in more detail below, term " mark " can refer to direct traget antibody, if coupling (namely physically connect) detectable material is on antibody, also can refer to indirect labelling antibody, this antibody by with other substance reaction directly marked.The example of indirect labelling comprises and detecting first antibody by fluorescent mark second antibody.

Sample can contact solid support or carrier as nitrocellulose, or can other solid supports of fixed cell, cell granulations or soluble protein or carrier.Then with upholder described in suitable buffer solution, the fingerprint gene-specific antibody process of detectable label is then used.And then solid support is washed, to remove unconjugated antibody by damping fluid second time.Finally can detect by the method for routine the amount being combined in and solid support marks.

" solid support or carrier " refers to can any support of conjugated antigen or antibody.The upholder known or carrier comprise glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified-cellulose, polyacrylamide and magnetite.The character of carrier is solvable to a certain extent, or insoluble for the object of the invention is.Described solid support material can have any possible node configuration, as long as the molecule of coupling can conjugated antigen or antibody.Therefore, supporting structure can be spherical, as pearl or columniform, as expressed in test tube, or the outside surface of rod.Preferred upholder comprises polystyrene bead.Those those skilled in the art will know other carrier that is suitable, that be used for binding antibody or antigen a lot, or can be determined by normal experiment.

A kind of method that can mark with detecting for the antibody of target protein is specifically connected with enzyme by this antibody, then (can refer to Voller for enzyme immunoassay (EIA), 1978, " The Enzyme Linked ImmunosorbentAssay (ELISA) ", Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.; Voller et al., 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa etc., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, the full content of each file of Tokyo. is hereby incorporated by).With the enzyme of antibodies by with suitable substrate reactions, be preferably chromogenic substrate.By by this way, detect the detectable chemical substance of generation as crossed spectrophotometry, fluorescent method or vision means.The enzyme that may be used for detectable label antibody includes but not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, δ-5-steroid isomeras, Alcohol Dehydrogenase from Yeast, alpha-phosphate glycerol dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.Described detection can complete by using colorimetric method, and the method adopts the chromogenic substrate of enzyme.Naked eyes can also be carried out by the degree of being reacted by enzyme-to-substrate with the standard substance of similar preparation and compare detection.

Detection can also be completed with any other immunization method various.Such as, utilize radiolabeled antibody or antibody fragment, likely by use radioimmunoassay (RIA) come testing goal albumen (see, such as, Weintraub, 1986, radioimmunoassay principle, 7th training course radioassay technology, Endocrine Society, is incorporated to its entirety herein by reference at this).Radio isotope (such as ¹²⁵i, ¹³¹i, ³⁵s or ³h) can by such as using gamma counter or scintillometer or being detected by radioautograph.

In addition, also fluorescent compound label antibody can be used.When fluorescently-labeled antibody is exposed to the light of suitable wavelength, just by the existence of fluoroscopic examination antibody.Wherein the most frequently used fluorescent labelling compound is fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, o-phthalaldehyde and fluorescamine.

Also can with send fluorescence metal detectable ground traget antibody, as ¹⁵²eu or other lanthanon.These metals can by metal chelating groups as diethylidene nitrilotriacetic (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA) be connected to antibody.

Can also by antibody coupling to chemiluminescent compound realizing detectability ground traget antibody.Detect the antibody that the light produced in chemical reaction process just can determine chemiluminescent labeling.Useful especially chemiluminescent labeling compound is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, stain Acridine ester, imidazoles, acridinium salt and barkite.

Similarly, bioluminescent compound can be used for marking antibody of the present invention.Noclilucence is a kind of chemoluminescence found in biosystem, and wherein catalytic protein improves the efficiency of chemiluminescence reaction.By detecting luminous existence to determine the existence of bioluminescent protein.Luciferin, luciferase and aequorin for marking the important bioluminescent compound of object.

In another embodiment, except antibody, molecule such as the aptamer of specific binding can be used in conjunction with biomarker.In another embodiment, biomarker spectrum can comprise detectable part in infectious agent (such as, lipopolysaccharides or viral protein) or its component.

In some embodiments, protein chip assay method (such as, biomarker system, Sai Fuji, Freemont, California) for detecting the biomarker characteristic value in biomarker spectrum.Also see, such as, Lin2004, Modern Pathology, 1-9; Li, 2004, Journal of Urology, 171,1782-1787; Wadsworth, 2004, Clinical Cancer Research, 10,1625-1632; Prieto, 2003, Journal ofLiquid Chromatography & Related Technologies 26,2315-2328; Coombes, 2003, ClinicalChemistry 49,1615-1623; Mian, 2003, Proteomics 3,1725-1737; Lehre etc., 2003, BJU International 92,223-225; And Diamond, 2003, Journal of the American Society forMass Spectrometry, 14,760-765.Its each be incorporated to herein with its entirety by reference at this.

In some embodiments, pearl assay method is for detecting the biomarker characteristic value in biomarker spectrum.The flow microsphere Array Method system (CBA) of BectonDickinson company is exactly a pearl assay method.CBA uses a series of particle and discrete fluorescence intensity to detect multiple soluble analyte simultaneously.CBA is combined with flow cytometry and creates multichannel and test.The system of Becton Dickinson company CBA, concrete example, as Becton Dickinson people anti-inflammatory agent box, based in the immunoassay of particle, uses flow cytometry to amplify the sensitivity technique soluble analyte of fluoroscopic examination.What every pearl in CBA provided specific proteins catches surface, be similar to an ELISA flat board, the hole of bag quilt separately.It is suspend, so that allow the compound detecting multiple small volume that the CBA of BD company catches pearl mixture.

In some embodiments, multiple analysis method describes at United States Patent (USP) 5981180 (calling in the following text " ' 180 patent "), by reference its entirety is incorporated to, especially for instruction universal method, pearl technology, system hardware and antibody test, and be used for detecting the biomarker of biomarker spectrum.In order to this analysis, the matrix of synthesis particulate, wherein said matrix comprises and is made up of the particulate of difference group.Often group particulate can have the molecule of thousands of different antibody capture reagent to be fixed on microparticle surfaces and be that color is just encoded, because be mixed with two kinds of fluorescence dyes of different amount.The ratio of two kinds of fluorescence dyes is often organize particulate to provide a different emmission spectrum, therefore identifies the particulate in Particle Swarm.U.S. Patent number 6268222 and 6599331 also all by way of reference entirety be incorporated to, the particulate especially for instruction mark carries out the various methods of multiple analysis.

5.4.3 other detection method used

In some embodiments, separation method may be used for the biomarker characteristic value detecting biomarker spectrum, like this with regard to a subset of biomarker in energy analytical specimen.Such as, analyzed biomarker can be the mRNA from cell extract.These cell extracts are fractionated, and only obtain nucleic acid biomarker, or these biomarkers can from a part for albumen total complement in sample, and these samples are by chromatographic technique fractionation.

The eigenwert of the biomarker of biomarker spectrum also can produce by using one or more methods as described below.Such as, nucleus magnetic resonance (NMR) wave spectrum, mass spectroscopy can be comprised, as electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/ (MS) ⁿsilicon (DIOS), second ion mass spectroscopy (SIMS), Quadrupole-time of flight mass spectrometry (Q-TOF), atmospheric pressure chemical ionization mass spectrum (APCI-MS), APCI-MS/MS, APCI-(MS) in (n is that an integer is greater than zero), laser desorption ionisation flight time mass spectrum (MALDI-TOF-MS), surface-enhanced laser desorb/ionization time of flight mass spectrometry (SELDI-TOF-MS), desorb/ionization ⁿ, atmospheric pressure photoionization mass spectrum (APPI-MS), APPI-MS/MS and APPI-(MS) ⁿ.Other mass spectrometry method can comprise, especially four poles, Fourier Transform Mass Spctrometry (FTMS) and ion trap.Other suitable method can comprise chemical extraction distribution, column chromatography, ion exchange chromatography, hydrophobic (anti-phase) liquid phase chromatography, isoelectrofocusing, one dimension polyacrylamide gel electrophoresis (PAGE), two dimensional polyacrylamide gel electrophoresis (2D-PAGE) or other chromatography, such as thin layer, gas phase or liquid phase chromatography, or its arbitrary combination.In one embodiment, described biological sample can first fractionation before application separation method.

In one embodiment, mastrix-assisted laser desorption ionization time of flight mass spectrum is for detecting the eigenwert of the biomarker in biomarker spectrum, wherein said biomarker is albumen or protein fragments, and they are ionized, and is evaporated to fixing upholder by laser radiation.Eigenwert is whether peak represents existence, and described peak represents these fragments in mass spectrographic pattern.Various laser desorption/ionization techniques is known in the art, can see Guttman et etc., 2001, Anal.Chem.73:1252-62 and Wei etc., 1999, Nature 399:243-246.Its entirety is incorporated to by each content by way of reference.

Mastrix-assisted laser desorption ionization time of flight mass spectrum can generate a large amount of information within the time of one relatively short.Added to by biological sample on different carrier, described biological sample or its subset are in conjunction with all biomarkers.Through or without the cell lysate of purifying or fractionation or sample, be low to moderate 0.5 μ L with volume and be applied directly to these carrier surfaces.Before being in application to carrier surface, can by lysate or sample concentration or dilution.Then for generating sample or the sample of laser desorption/MALDI-MS, the time used only needs short 3 hours.

5.4.4 data analysis algorithm

The express spectra of T-MSC biomarker is the reference factor distinguishing clinical rank and non-clinical rank T-MSC.The identity of these biomarkers and corresponding function (such as, expressing) thereof can be used for setting up one or more decision ruless.One or more decision ruless that particular data analytical algorithm is set up, can distinguish clinical rank and non-clinical rank T-MSC.Once use these representative data analytical algorithms or other technology as known in the art to set up decision rules, these decision ruless will be used for being two or more rank (such as clinical rank and non-clinical rank) by T-MSC heap sort.This realizes by decision rules being applied to the biomarker spectrum obtained from cell culture.Because of a little, such decision rules has huge value when definition of T-MSC.

In certain embodiment, there is provided herein a kind of evaluation method for comparing the biomarker spectrum obtained to be measured and compared with control cells culture.In some embodiments, the often kind of biomarker spectrum obtained from control group or cell culture to be measured forms the wherein eigenwert multiple biomarker spectrum.In some embodiments, this comparison passes through: (i) uses the biomarker of control group to set up a kind of decision rules; (ii) described decision rules is applied to the biomarker spectrum of cell culture to be measured.Like this, the decision rules applied in some embodiments of the present invention is used for determining to treat that cell is clinical rank or non-clinical rank.In other embodiments, control group is BM – MSC.

In certain embodiments of the present invention, when application decision rule qualification cell culture to be measured is clinical rank T-MSC, can be used as treatment.If qualification result finds the clinical rank of right and wrong, so treatment can not be used as.

The modification of 5.5T-MSC

A kind of mescenchymal stem cell production a group immune suppression function of modification is provided to obtain the method for the MSC group of the improvement promoted herein.Described MSC has following characteristics: (i) cell expressing group-1 mark containing >95%; (ii) cell expressing group-2 mark containing >80%; (iii) cell expressing group-3 mark containing <5%; (iv) IL-10 and TGF β is expressed; (v) cell expressing IL-6, IL-12 and TNF α containing <2%; (vi) all groups-1 marks of the cell coexpression containing <0.001%.Described here group-1 mark is: CD73, CD90, CD105, CD146, CD166 and CD44; Group-2 mark is: CD13, CD29, CD54 and CD49E; Group-3 mark is CD45, CD34, CD31 and SSEA4; Group-4 mark is: OCT4, NANOG, TRA-1-60 and SSEA4.

A kind of method promoting the immune suppression function of T-MSC by improving AIF expression is provided herein.In one embodiment, described method comprises the down-regulated expression of PIF.In one embodiment, described method comprises IL6, IL12, TNF α, RAGE and other PIF down-regulated expression at T-MSC.In one embodiment, described method comprises the up-regulated of TGF β and IL-10 at T-MS.

In some embodiments, described method comprises heredity and the epigenetic modification of T-MSC, and described modification is known in the art.In some embodiments, genetic modification and epigenetic modification include but not limited to knock out, children purpura nephritis (" shRNA "), small non-coding RNA (" miRNA "), non-coding RNA (" ncRNA "), morpholino oligonucleotides, bait RNA, DNA methylation regulation and control, histone methylated regulation and control, Translational repression and/or antibody closes.In some embodiments, by transposon, toll sample receptors ligand or small numerator modified MSC.

In some embodiments, any element of small molecules targeting IL-6 signal path is used.In some embodiments, RN SYP target includes but not limited to gp130, STAT3, Cathepsin S, NFkappaB and IRF5.In some embodiments, make the IL-12 of T-MSC express in the following manner to reduce: prostaglandin E2 signal path activates; In born of the same parents, cyclic amp (cAMP) and cAMP agonist include but not limited to that the level of forskolin, Toxins,exo-, cholera, β 1 and β 2 adrenal epinephrine acceptor rises; NF-κ B Rel-B signal path is suppressed; Use apoptotic cell process T-MSC; Use phosphatidylserine process T-MSC; Use butyrates process T-MSC; The triptolide (i.e. Minnelide) of triptolide or Radix Tripterygii Wilfordii extract or synthesis is used to process T-MSC.

In some embodiments, the technology known by recombination field can be utilized to modify MSC makes it express a certain mark.In some embodiments, the nucleotide sequence by transfection coded markings modifies MSC.Described mark can be inserted into suitable expression vector, namely containing the carrier transcribing and translate necessary element to the encoding sequence inserted.Necessary transcribe and translate element and also can exist.Regulatory region and enhancer element can be various sources, natural in synthesis.Various host-vector system can be used for expressing described mark.These include but not limited to the mammalian cell system infecting virus (as virus, vaccinia virus, adenovirus etc.); Infect the insect cell system of virus (as baculovirus); Microorganism is as comprised the bacterium of the yeast of yeast vector and transformed bacteriophage, DNA, plasmid DNA, cosmid DNA; By transforming the stable cell lines of selectable markers.Advantage and the feature of vector expression element are different.According to the host-vector system used, many suitable transcribing and translate any one in element can be used.

Once the carrier of composite coding appropriate flags, can by the emerging object vector of sense or transfection MSC.

Can use standard method that object nucleotide sequence is introduced MSC.Polynucleotide can be introduced host cell by any known method for transformation, comprise as polynucleotide being packaged into virus and by viral transduction to host cell, or directly take in polynucleotide.Mammals transforms (i.e. transfection), directly takes in by calcium phosphate precipitation (see Graham and Van derEb, 1978, Virol.52:546) or the virus of known modification.Recombination of polynucleotide is introduced cell, especially the method for mammalian cell, comprise the transfection of dextran mediation, the transfection of calcium phosphate mediation, polybrene mediation transfection, protoplast fusion, electroporation, polynucleotide are packaged in liposome, by polynucleotide under the microscope direct injection to nucleus.These methods are well-known to those skilled in the art.

In preferably implementing at one, the stable cell lines containing object carrier is used for high flux screening.The preparation of this stable cell lines comprises optionally labeled vector by introducing, and allows cell at perfect medium growth 1-2 days, then makes Growth of Cells in selective medium.Selected marker in recombinant plasmid is used for selection, and allows the cell of stable this recombinant plasmid of insertion in karyomit(e) long-living, carries out formation clone and increases as clone.

Operable selective system has multiple, include but not limited to herpes simplex virus thymidine kinase (see Wigle etc., 1977, Cell 11:223), xanthoglobulin-guanine phosphoribosyl base enzyme (see Szybalska and Szybalski, 1962, Proc.Natl.Acad.Sci.USA 48:2026) and adenine phosphoribosyl transferase be (see Lowy, et al., 1980, Cell 22:817), these genes can knock out at tk respectively, hgprt ^-knock out or use in cell that aprt knocks out.In addition, can by metabolic antagonist resistance with electing, such as, dhfr can produce methotrexate resistance (see Wigler etc., 1980, Natl.Acad.Sci.USA 77:3567; O'Hare, etc., 1981, Proc.Natl.Acad.Sci.USA 78:1527); Gpt can produce the resistance (see Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA 78:2072) to mycophenolic acid; Neo can produce the resistance (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1) to aminoglycoside G418; Hygro can produce resistance to Totomycin (see Santerre etc., 1984, Gene 30:147).

5.6 stem cell collection compositions

Stem cell collection composition can comprise any physiologically acceptable solution being suitable for collection and/culturing stem cells, such as salts solution (Kreb solution, Eagle solution, 0.9%NaCl solution etc. as phosphate buffered saline buffer, Kreb solution, improvement) and substratum (as DMEM, H.DME etc.), etc.

Stem cell collection composition can comprise one or more components of protection stem cell, namely from collecting between cultivation, prevents stem cell from avoiding death, or postpones the death of stem cell, reduces the quantity of the stem cell of dead cell group, etc.Such assembly can be, such as inhibitors of apoptosis (such as Caspase inhibitors or jnk inhibitor); Vasodilator (such as magnesium sulfate, antihypertensive drug, atrial natriuretic peptide (ANP), thyroliberin, corticotropin releasing hormone, Sodium Nitroprusside, hydralazine, Triphosaden, adenosine, indomethacin or magnesium sulfate, phosphodiesterase inhibitor etc.); Downright bad inhibitor (such as, 2-(1H-indol-3-yl)-3-penta amino-maleimide, PDTC or clonazepam); TNF-alpha inhibitor; Take oxygen perfluoro-carbon (as PERFLUBRON, PFDB etc.).

Stem cell collection composition can comprise one or more tissue degradation enzymes, such as metalloprotease, serine protease, neutral protease, rnase or deoxyribonuclease, etc.This fermentoid includes but not limited to collagenase (such as collagenase I, II, III or IV, the collagenase etc. from clostridium histolyticum), Dispase, thermolysin, elastoser, trypsinase, release enzyme, Unidasa, etc.

Stem cell collection composition can comprise the microbiotic of sterilization or antibacterial significant quantity.In some nonrestrictive embodiment, described microbiotic is macrolide (such as tobramycin), cynnematin (such as Cephalexin Monohydrate Micro/Compacted, Cephradine, cephalofruxin, Prozef, cefaclor, Cefixime Micronized or S 578), clarithromycin, erythromycin, penicillin (such as penicillin v) or quinolones (such as Ofloxacine USP 23, Ciprofloxacin or norfloxicin), tsiklomitsin, Streptomycin sulphate etc.

Stem cell collection composition can also comprise one or more following compounds: adenosine (about 1mM to about 50mM); D-Glucose (about 20mM to about 100mM); Magnesium ion (about 1mM to about 50mM); Molecular weight is greater than 20000 daltonian macromole.In one embodiment, the content of following material is enough to maintain endothelium integrity and cytoactive, such as, and synthesis or natural colloid, polysaccharide such as dextran or polyoxyethylene glycol, about 25g/l to about 100g/L, or about 40g/L to about 60 grams/L; Antioxidant, such as butyl hydroxyanisole, butylhydroxy toluene, gsh, vitamins C or vitamin-E about 25 μMs to about 100 μMs; Reductive agent, the N-acetylcystein of such as about 0.1mM to about 5mM; Calcium ion is stoped to enter the medicament of cell, such as verapamil, about 2 microns to about 25 microns; Anti-coagulant.In one embodiment, the content of following material is enough to help prevent residual blood coagulation, such as heparin or r-hirudin, about 1000 units/l to about 100000 units/l; Or such as, containing guanamprazine compound, guanamprazine, ethylisopropyl base amiloride, hexa-methylene amiloride, dimethyl amiloride or isobutyl-amiloride, about 1.0 μMs to about 5 μMs.

5.7 use T-MSC immunomodulatory

The adjustment of one or more immunologic cellular activity (such as, downward cell proliferation, reduction cell survival, block cell migrate to by inflammation sites, lower the homeostatic function of recovery that cell promotes or extend the ability of inflammation, raising cell promotion health tissue or organ) is provided herein.This to be contacted with multiple T-MSC or iT-MSC by immunocyte (or cell mass) and realizes.In one embodiment, immunity moderation response method, comprise multiple immunocyte contact with multiple T-MSC or iT-MSC the sufficiently long time with make T-MSC or iT-MSC can detect ground Immunosuppression react.Wherein, in mixed lymphocyte reacion (MLR), T-MSC or iT-MSC can detect ground suppressor T cell propagation.

Because the MSC in BM-MSC or other adult tissue source has been used to treat many autoimmune disorders, BM-MSC has also been applied to tissue repair by restriction inflammation, secretion somatomedin, secretion protective factors and replacement damaged tissue.As shown in example below, T-MSC has more excellent immune suppression function than BM-MSC.Therefore, T-MSC can be applied to all areas and the disease of current BM-MSC application.

For immunoregulatory T-MSC or iPS-MSC can be derivative from the multipotential stem cell system of embryonic stem cell line or induction or obtain respectively.For immunoregulatory T-MSC or iPS-MSC can also from the species identical with the immunocyte that activity will be conditioned; Or from the species different from the immunocyte that activity will be conditioned.

In the context of present method, " immunocyte " refers to cell any in immunity system, particularly T cell and NK (natural killer) cell.Therefore, in the different embodiments of present method, T-MSC contacts with multiple cell.Wherein said multiple cells, are or comprise multiple T cell (such as, multiple CD3 ⁺t cell, CD4 ⁺t cell and/or CD8 ⁺t cell) and/or natural killer cell.In the context of method, " immunne response " can be immunocyte usually can any reaction of perceptible stimulus to what sent by immunocyte, such as, to the reaction that antigen occurs.In various embodiments, immunne response can be T cell propagation response exotic antigen.Wherein, T cell such as CD3 ⁺t cell, CD4 ⁺t cell and/or CD8 ⁺t cell; Exotic antigen such as in autoimmune disorder, blood transfusion or the antigen of graft.Immunne response also can be included in the propagation of the T cell in graft.Immunne response also can be the maturation of any activity of natural killer cell, dendritic cell, etc.Immunne response also can be active one or more immunocytes specific or systematic effect in local, tissue or organ.Immunne response can be graft versus host disease (GVH disease), inflammation, formation inflammation are correlated with scar tissue, autoimmune syndrome (such as rheumatoid arthritis, type i diabetes, systemic lupus erythematous etc.), etc.

" contact " in this context comprising: T-MSC cell is placed on single container (such as culture dish, flask, bottle etc.) or in vivo, such as same individuality (such as Mammals, such as people) together with immunocyte.In a preferred embodiment, the time of contact is sufficiently long, and T-MSC cell and immunocyte quantity abundant so that the change of the immunologic function of immunocyte is detectable.More preferably, in various embodiments, described contact is enough to Immunosuppression function (as T cell propagation response antigen example), and does not exist compared with T-MSC, and T-MSC at least suppresses the immunologic function of 50%, 60%, 70%, 80%, 90% or 95%.This suppression in vivo can measure in vitro, that is, can infer the T-MSC of specific quantity suppression degree to some immunocytes in recipient's body in test in vitro.

In some embodiments, the invention provides use T-MSC and regulate multiple immune response of one or more type of immune cells or the method for activity in vitro.T-MSC contacts with multiple immunocyte and can comprise T cell and immunocyte at same entity space, like this, and a wherein part of at least multiple T-MSC and the wherein part interaction of multiple immunocyte; Maybe can comprise, the substratum from T-MSC or immunocyte culture contacts with other cell type (substratum such as, obtained from the culture of T-MSC and the isolating immune cells of Eddy diffusion this substratum).An embodiment, contact is a mixed lymphocyte reacion (MLR).

Such as, such contact can occur in the testing apparatus that is detected T-MSC immunomodulatory (as immunosuppression) degree.Such testing apparatus can be, such as mixed lymphocyte reacion (MLR) or regression analysis.Mixed lymphocyte reacion and regression analysis are known in the art, can see such as: Schwarz, " The MixedLymphocyte Reaction:An In Vitro Test for Tolerance, " J.Exp.Med.127 (5): 879-890 (1968); Lacerda etc., " Human Epstein-Barr Virus (EBV)-Specific Cytotoxic TLymphocytes Home Preferentially to and Induce Selective Regressions of AutologousEBV-Induced B Lymphoproliferations in Xenografted C.B-17Scid/Scid Mice, " J.Exp.Med.183:1215-1228 (1996).In one preferred embodiment, mixed lymphocyte reacion multiple T-MSC and multiple immunocyte (as lymphocyte, such as CD3 ⁺cD4 ⁺and/or CD8 ⁺t lymphocyte) contact in carry out.

The immunosuppression capability of the fixed multiple T-MSC of this MLR Ke Lai Measuring.Such as, multiple T-MSC can be detected in MLR, this MLR comprise by, such as the ratio of about 10:1:2 is in conjunction with CD4 ⁺or CD8 ⁺t cell, dendritic cell (DC) and T-MSC, wherein utilize dyestuff, such as, distribute the CFSE (as an example) entering daughter cell, dye to this T cell, and wherein allow this T cell to breed about 6 days.If the T cell of 6 days is bred when there is T-MSC, compared with breeding with T cell when there is DC and there is not T-MSC, can detect it is reduce, then the plurality of T-MSC is subject to immunosuppression.In such mixed lymphocyte reacion, T-MSC can thaw, and also can collect from culture.About 10000 T-MSC are resuspended in (RPMI1640,1mM HEPES damping fluid, microbiotic and 5% PHS) in 100ml substratum, and make it be attached at the bottom of ware 2 hours.Utilize immunomagnetic beads from full periphery blood monocyte separation of C D4 ⁺and/or CD8 ⁺t cell.These cells are CFSE dyeing, and 100000 T cell (only add CD4 altogether ⁺, only add CD8 ⁺, or the CD4 of equivalent ⁺and CD8 ⁺t cell) be added into each hole.The volume of each hole liquid is mended to 200 μ l, and allows to carry out mixed lymphocyte reacion.

In one embodiment, therefore, the invention provides a kind of method of Immunosuppression cell response, wherein, in mixed lymphocyte reacion, multiple immunocyte contact with multiple T-MSC the sufficiently long time with make T-MSC can detect ground suppressor T cell propagation.

The T-MSC group's never obtained with embryonic stem cell line can be different in the ability of immunity moderation cytoactive, such as can be different in the ability of or propagation or NK cytoactive active in suppressor T cell.Therefore, the immunosuppression capability of the surely specific T-MSC group of use Qian Measuring is needed.This kind of ability is passable, and such as, sample Lai Measuring by detecting this amnion-derived attached cell group in MLR or regression analysis is fixed.In one embodiment, perform MLR with described sample and in described analysis, measure the immunosuppression degree that this T-MSC group causes.Then, can by this immunosuppression degree owing to sampled T-MSC group.Therefore, this MLR can be used as a kind of method measuring the absolute and relative ability of specific T-MSC group's Immunosuppression function.MLR Ginseng number can be how auspicious change, with the immunosuppression capability providing more data or the Hao Di Measuring of Geng to determine T-MSC sample.Such as, because the immunosuppression that T-MSC produces seems to increase roughly in the mode proportional with the quantity of T-MSC that exists in this analysis, therefore, in one embodiment, can with the T-MSC group of two or more quantity (such as each reaction 1 × 10 ³, 3 × 10 ³, 1 × 10 ⁴and/or 3 × 10 ⁴individual T-MSC) perform this MLR.In this analysis, the relative populations of T-MSC and T cell can also be changeable.Such as, although relatively a large amount of T-MSC or T cell can be used, in this analysis, T-MSC and T cell can exist by any ratio (such as about 100:1 is to about 1:100, preferably about 1:5).

The present invention also provides the method for the multiple activity using the reaction of T-MSC immunity moderation in vivo or regulate one or more immunocytes.T-MSC and immunocyte passable, such as contact in the individuality of the recipient as multiple T-MSC.When this contact is carried out in individuality, in one embodiment, this contact occurs in T-MSC (namely, T-MSC not derives from this individuality) and is between endogenic multiple immunocyte concerning this individuality.In a particular embodiment, the immunocyte of this individuality is CD3 ⁺t cell, CD4 ⁺t cell, CD8 ⁺t cell and/or natural killer cell.

That the immunosuppression using T-MSC to bring causes for any situation or that worsen or relevant or less desirable immune response is good.The immunomodulatory of T-MSC mediation, as immunosuppression, will, be such as conducive to suppressing unsuitable immune response, this immune response is caused by individual immunity system counter its tissue one or more.Therefore, the invention provides the method for a kind of Immunosuppression reaction, wherein, described immune response is autoimmune disorder, as systemic lupus erythematosus, diabetes, rheumatic arthritis or multiple sclerosis.

Multiple T-MSC can occur in recipient's individuality with the contacting of multiple immunocytes of one or more types, and this recipient is grafted or transplants one or more tissues, or these cells are as annexation in the lump grafting or transplanting.These tissues can be, such as marrow or blood, organ, special tissue (as dermatoplasty), complex tissue (as graft comprises two or more tissue) etc.In this regard, T-MSC may be used for the one or more immune responses suppressing one or more immunocyte, and described immunocyte is included in recipient's individuality or transplanted tissue or grafting tissue or two tissues.Before, during and/or after this contact can occur in grafting or transplanting.Such as, T-MSC process can be given when transplanting or grafting.Or optionally, also can transplanting or grafting give T-MSC process, such as about transplanting or grafting before 1,2,3,4,5,6,7 day.Preferably, before immunoreactive any detectable signal or symptom occur, multiple T cell and multiple T-MSC will be made to contact.This detectable signal or symptom can be the individual or transplanted tissue of recipient or grafting, as detectable signal or the symptom of graft versus host disease (GVH disease) or detectable inflammation.

In another embodiment, the intraindividual contact mainly T-MSC of external source and endogenous progenitor cell or stem cell, as the immunocyte that endogenous progenitor cell or differentiation of stem cells become.Such as, be in the individuality that part or all of immunologic purging therapy or medullary cell are removed, T-MSC can be accepted in conjunction with the stem cell of one or more types or the progenitor cell appurtenant as cancer therapy.Such as, T-MSC can in conjunction with multiple CD34 ⁺cell, as CD34 ⁺hemopoietic stem cell.These CD34 ⁺cell can be such as carry out self-organization, such as periphery blood, Cord blood, placental blood or marrow.These CD34 ⁺cell can from the above separate tissue, or the partially purified sample (such as from the white corpuscle of umbilical cord) of whole tissue of originating (such as, whole Cord blood or marrow) or tissue, and these cells can be combined with T-MSC.

This T-MSC can be applied to individuality, and wherein known in this T-MSC and this individuality or the ratio of immunocyte (such as T cell) quantity expected is from about 10:1 to about 1:10, preferably about 1:5.But in a non-limiting embodiment, multiple T-MSC can be by, about 10,000:1, about 1,000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1,000 or about 1:10, the ratio of 000 is applied to individuality.Usually, about 1 × 10 can be used ⁵to about 1 × 10 ⁸individual T-MSC/kg (recipient's body weight), preferably about 1 × 10 ⁶to about 1 × 10 ⁷individual T-MSC/kg (recipient's body weight), to produce immunosuppression.In various embodiments, the T-MSC being applied to individuality or object comprises at least, about or no more than, and 1 × 10 ⁵, 3 × 10 ⁵, 1 × 10 ⁶, 3 × 10 ⁶, 1 × 10 ⁷, 3 × 10 ⁷, 1 × 10 ⁸, 3 × 10 ⁸, 1 × 10 ⁹, 3 × 10 ⁹individual T-MSC or more.

T-MSC also can use together with the stem cell of one or more Second Types, as mesenchymal stem cells MSCs.The stem cell of this Second Type can be administered to individuality, the ratio 1:10 to about 10:1 according to appointment of they and T-MSC.

Contact with immunocyte for the ease of T-MSC, this T-MSC can be applied to individuality by any approach being enough to make this T-MSC and immunocyte contact with each other.Such as, this T-MSC can pass through, such as intravenously, intramuscular, intraperitoneal, intraocular, parenteral, in sheath or the approach directly entering organ (such as pancreas) be applied to individuality.Use for external, T-MSC can make pharmaceutical preparation and perform.

Described IM can also comprise and add one or more immunosuppressor, particularly environment in vivo.In one embodiment, multiple T-MSC contacts multiple immunocyte in individual body, and composition comprises immunosuppressive drug is administered to individuality.Immunosuppressive drug is known in the art, includes but not limited to anti-φt cell receptor antibody (as mono-clonal or polyclonal antibody, or antibody fragment or derivatives thereof), anti-IL-2 receptor antibody (such as, basiliximab or Zenapax anti-φt cell receptor antibody (such as Muromonab-CD3), azathioprine, reflunomide, ciclosporin, tacrolimus, mycophenlate mofetil, sirolimus, calcineurin inhibitors etc.In an embodiment, described immunosuppressor is the neutralizing antibody for macrophage inflammatory protein (MIP)-1 α and MIP-1 β.

The preservation of 5.8T-MSC and/or T-MSC-DL

T-MSC and/or T-MSC-DL can be saved, namely by they prolonged storage, or the necrocytosis that suppression is caused by such as apoptosis or necrosis.Compound can be used to preserve T-MSC and/or T-MSC – DL, and these compounds comprise inhibitors of apoptosis and downright bad inhibitor.In one embodiment, the invention provides a kind of method of preserving population of stem cells, wherein, the stem cell collection composition of population of stem cells contact containing inhibitors of apoptosis, the amount that wherein said inhibitors of apoptosis exists exists to be enough to reduce or stop population of stem cells apoptosis (compared with not contacting the cell mass of this inhibitors of apoptosis) with the time existed.In a concrete embodiment, inhibitors of apoptosis is Caspase inhibitors.In another embodiment, inhibitors of apoptosis is jnk inhibitor.In further embodiment, jnk inhibitor does not does not regulate and control propagation and the differentiation of described stem cell.In another embodiment, described cell harvesting composition comprises described inhibitors of apoptosis and is separated described in phase and takes oxygen perfluoro-carbon.In another embodiment, described cell harvesting composition comprises described inhibitors of apoptosis and is contained in described in emulsion and takes oxygen perfluoro-carbon.In another embodiment, this cell harvesting composition additionally comprises emulsifying agent, such as Yelkin TTS.In another embodiment, described inhibitors of apoptosis and described perfluoro-carbon, when contacting this cell, are between about 0 DEG C to about 25 DEG C.In another more specifically embodiment, described inhibitors of apoptosis and described perfluoro-carbon, when contacting this cell, are between about 2 DEG C to 10 DEG C or between about 2 DEG C to about 5 DEG C.In another more specifically embodiment, described contact performs during the described cell mass of transfer.In another more specifically embodiment, described contact is freezing and perform during melting described population of stem cells.

In another embodiment, the invention provides a kind of method of preserving T-MSC and/or T-MSC – DL group, wherein, the composition of population of stem cells contact inhibitors of apoptosis and preservation organ, wherein, the amount that inhibitors of apoptosis exists was enough to lower the apoptosis (not contacting inhibitors of apoptosis compared to population of stem cells) reduced with stoping population of stem cells with the time.

Usually, collect at T-MSC and/or T-MSC-DL, between enrichment and separation period, preferably minimize or eliminate the cellular stress because anoxic and mechanical stress cause.Therefore, in another embodiment of the method, between stem cell or population of stem cells preservation period, stem cell or population of stem cells are exposed in low-oxygen environment, are no more than 6 hours to their collection, enrichment with the time be separated.Wherein, low-oxygen environment refers to that the concentration degree of oxygen is lower than mesectic concentration.In one more specifically embodiment, stem cell is during preservation exposed to low-oxygen environment and is less than 2 hours.In another more specifically embodiment, population of stem cells is during preservation exposed to low-oxygen environment and is less than 1 hour or is less than 30 minutes, or is not exposed to low-oxygen environment during collecting, during enrichment or between separation period.In another more specifically embodiment, population of stem cells is not exposed to shearing stress between collection, enrichment or separation period.

T-MSC and/or T-MSC – DL can contain the small vessels (such as ampulla) as freezen protective substratum by freezen protective.Suitable freezen protective substratum includes but not limited to, comprises following substratum: such as, growth medium or cell cryopreservation substratum, and such as, the cell cryopreservation substratum be purchased, as C2695, C2639 or C6039 (Sigma).Freezen protective substratum preferably comprises the dimethyl sulfoxide (DMSO) (DMSO) of concentration such as about 10% (V/V).Freezen protective substratum can comprise other reagent, such as methylcellulose gum and/or glycerine.T-MSC and/or T-MSC – DL is in process of cryopreservation, and preferred speed of cooling is about 1 DEG C/min.Preferred freezen protective temperature is about-80 DEG C to about-180 DEG C.Before thawing uses, preferably, about-125 DEG C to about-140 DEG C frozen cells can be transferred in liquid nitrogen.In some embodiments, such as, once the temperature of ampulla reaches to about-90 DEG C, they are transferred to containing liquid nitrogen storage region.During the cell thawing of freezen protective preferably at the temperature of 25 DEG C to about 40 DEG C, preferred temperature about 37 DEG C.

T-MSC and/or T-MSC-DL of 5.9 freezen protective

T-MSC and/or T-MSC-DL disclosed herein can be saved, and such as freezen protective for future use.Cell Cryopreservation, the method for such as stem cell is known in the art.T-MSC and/or T-MSC-DL can conveniently be administered to individual method preservation with a kind of.Such as, provide T-MSC and/or T-MSC-DL be contained in container herein, this container is applicable to medical use.This container can be, such as aseptic plastics bag, flask, tank, or other easily can dispose the container of T-MSC and/or T-MSC-DL.Such as, described container can be blood bag or other plastics, be suitable for the medically acceptable bag of acceptor intravenously conveying liquid.This container preferably allows freezen protective population of stem cells.T-MSC and/or T-MSC-DL of freezen protective can comprise T-MSC and/or T-MSC-DL from a contributor or from multiple contributor.T-MSC and/or T-MSC-DL can mate with the complete HLA of expection recipient, or partially or completely HLA is unmatched.

In another particular embodiment of the invention, described container is bag, bottle or tank.In one more specifically embodiment, described bag is aseptic plastic bag.In one more specifically embodiment, described bag is applicable to, allows or contributes to T-MSC and/or T-MSC-DL intravenous administration.This sack can comprise multiple chamber or room, and before administration or period, described chamber or room are interconnected, and mix mutually to allow this cell and one or more other solution (such as medicine).In another particular embodiment of the invention, this composition comprises one or more compounds, promotes the population of stem cells cryopreservation of combination.In another particular embodiment of the invention, T-MSC and/or T-MSC-DL is comprised in the acceptable aqueous solution of physiology.In one more specifically embodiment, the acceptable aqueous solution of this physiology is 0.9%NaCl solution.In another particular embodiment of the invention, the population of stem cells of hES-MSC and recipient is HLA coupling.In another particular embodiment of the invention, the population of stem cells of described combination comprises hES-MSC, and these hES-MSC population of stem cells HLA that is at least part of and recipient mates.

5.10T-MSC is divided into various kinds of cell system

T-MSC can be divided into various kinds of cell system, comprises Neuronal lineage cell or neurone or adipocyte or myocyte or inoblast or scleroblast or chondrocyte.Unless stated otherwise, T-MSC can be inoculated in bag by gelatin, collagen protein, fibronectin, matrigel, ln, vitronectin or the Tissue Culture Plate gathering (Methionin).T-MSC can with concentration 1 × 10 ³cell/cm ²to 1 × 10 ⁴cell/cm ²be seeded to serum free medium or the blood serum medium containing FBS or ABHS.Can by following wherein a kind of method differentiation according to the above condition inoculation T-MSC.

In one embodiment, add 1-50ng/ml epithelical cell growth factor (EGF) (best is 10ng/ml) containing 1-50ng/m FGF-2m (FGF-2) (best is 10ng/ml) in the substratum that T-MSC can break up and add 0.5-5ng/ml platelet derived growth factor (PDGF) (best is 1ng/ml).Every 2-3 days replaced medium, and harvested cell after 2-4 week, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-2 × 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC is seeded in the culture plate wrapped by poly-L-Orn and ln, can be divided into Neuronal lineage cell.Dividing of T-MSC is divided into 3 stages.Stage 1:1-50ng/ml FGF-2 (the best is 10ng/ml) and 1-50ng/ml EGF (best is 10ng/ml), makes hMSCs break up towards neurocyte direction.Stage 2:10-200ng/ml Sonic hedgehog (SHH) (the best is 100ng/ml), 1-50ng/ml FGF-8 (people) (best is 10ng/ml) and 50-500 μM of AAP (the best is 200 μMs), start to break up to midbrain.The neurotrophic factor (GDNF) (best is 50ng/ml) that stage 3:5-500ng/ml is glial cell derived and 50-500 μM of AAP (the best is 200 μMs), with differentiation-inducing and ripe dopaminergic neuron.Each stage needs a week, and between each stage, uses pancreatin or TrypLE/ Dispase to dissociate AC to go down to posterity.Supplement somatomedin and every 2 days replaced medium every day.Expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶– 4x 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC (Gibco) can be divided into Neuronal lineage cell in neural matrix substratum, this substratum comprises 0.25 × B-27 fill-in, add 10-200ng/ml Sonic hedgehog (SHH) (best is 100ng/ml), add 1-50ng/ml FGF-8 (mouse) (best is 10ng/ml), add 1-200ng/ml FGF-2 (best is 50ng/ml).At the 6th day and the 12nd day collecting cell, not replaced medium in the meantime.Expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶– 4x 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC can through two stage differentiated be Neuronal lineage cell.Stage 1:T-MSC cultivates 48-72 hour in the plasma-free DMEM medium containing 2mM glutamine, 1-20U/ml (best is 12.5U/ml) nystatin, N2 fill-in, 2-50ng/ml (best is 20ng/ml) FGF-2m (FGF-2) and 1-50ng/mL EGF (best is 10ng/ml).Stage 2: cell is containing the glial cell derived neurotrophic factor (GDNF of B27 fill-in, 0.1-10mM (best for 1mM) dibutyryl cyclic adenosine monophosphate (dbcAMP), 3-isobutyl-1-methylxanthine (IBMX), 10-500 μM (the best is 200 μMs) xitix, 1-100ng/ml BDNF (best is 50ng/ml), 1-50ng/ml; the best is 10ng/ml), 0.2-10ng/ml Transforming growth factor-β3 (TGF-β 3; the best is 2ng/ml), 0.05-5 μM all-trans retinoic acid (RA, the best is 0.1 μM).Each stage needs a week, and between each stage, uses pancreatin or TrypLE/ Dispase to dissociate AC to go down to posterity.Every 2 days replaced medium, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4 × 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC can be cultivated and be broken up with induced osteogenesis.T-MSC will in the low sugar DMEM substratum containing 10%FCS, 1-150uM (the best is 80 μMs), 2-phosphoric acid xitix, 0.5-5 μM (the best is 1 μM) dexamethasone and 1-100mM (best is 20mM) beta-glycerophosphate.Every 2-3 days replaced medium, after 2 weeks, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4x 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC can be cultivated to be induced to differentiate into adipocyte.T-MSC will cultivate in the low sugar DMEM substratum containing 20%FCS, 1-10 μ g/ml (the best is 5 μ g/ml) Regular Insulin, 0.5-10 μM (the best is 2 μMs) dexamethasone, 0.1-1mM (best is 0.5mM) isobutyl methylxanthine and 1-100 μM of (the best is 60 μMs) indomethacin.Every 2-3 days replaced medium, after 4 weeks, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4 × 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC can be cultivated to be induced to differentiate into chondrocyte.T-MSC will with coccoid growth in the DMEM in high glucose substratum containing 0.5-10mM (best is 1mM) Sodium.alpha.-ketopropionate, 0.05-1mM (best is 0.1mM) 2-phosphoric acid xitix, 0.05-1 μM (the best is 0.1 μM) dexamethasone, 0.2-2% (the best is 1%) ITS and 1-50ng/ml (best is 10ng/mL) TGF-β 3.Every 2-3 days replaced medium, after 20 days, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4 × 10 ⁶neurocyte unit lineage.

In another embodiment, T-MSC can be cultivated to be induced to differentiate into sarcoplast.T-MSC cultivates in the low sugar DMEM substratum containing 10%FBS, 1-20 μM of (the best is 10 μMs) U-18496 and 1-50ng/ml (best is 10ng/ml) basic FGF.After 24 hours, the flesh inducing culture that becomes containing 10%FBS with 1-50ng/ml (best is 10ng/ml) basic FGF replaces DMEM substratum.Every 2-3 days replaced medium, after 2 weeks, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4 × 10 ⁶neurocyte unit lineage.

In another people's embodiment, T-MSC can be cultivated to be induced to differentiate into inoblast.T-MSC cultivates in the DMEM substratum containing 10%FBS, 50-200ng/ml (best is 100ng/ml) recombinant human Connective Tissue Growth Factor (CTGF), 1-100 μ g/ml (the best is 50 μ g/ml) xitix, and this substratum is used to cultivate hMSCs's.Every 3-4 days replaced medium, after 4 weeks, expected volume is every 1 × 10 ⁶t-MSC produces 0.5 × 10 ⁶-4 × 10 ⁶neurocyte unit lineage.

Utilize any differentiation method to include but not limited to from the derivative all cells system of T-MSC and cell type, be above-mentionedly called as T-MSC-DL all method.

5.11 pharmaceutical preparation

In one embodiment, provide a kind of pharmaceutical composition here, wherein, the T-MSC comprising treatment significant quantity and the carrier pharmaceutically all accepted.

Described pharmaceutical composition can comprise any amount of T-MSC and/or T-MSC-DL.Such as, the T-MSC of 1 unitary dose can comprise, and in various embodiments, about at least, or is no more than 1 × 10 ⁵, 5 × 10 ⁵, 1 × 10 ⁶, 5 × 10 ⁶, 1 × 10 ⁷, 5 × 10 ⁷, 1 × 10 ⁸, 5 × 10 ⁸, 1 × 10 ⁹, 5 × 10 ⁹, 1 × 10 ¹⁰, 5 × 10 ¹⁰, 1 × 10 ¹¹or more T-MSC and/or T-MSC-DL.

Pharmaceutical composition disclosed herein comprises cell mass, and these cell masses comprise the viable cell (namely, in cell mass, the cell of at least 50% has function or survival) of 50% or more.Preferably, in cell mass, the cell of at least 60% is alive.More preferably, in pharmaceutical composition, in cell mass, the cell of at least 70%, 80%, 90%, 95% or 99% is alive.

Pharmaceutical composition disclosed herein comprises one or more compounds, is such as conducive to the compound transplanted, such as, and anti-φt cell receptor antibody, immunosuppressor etc.; Also can be stablizer, such as albumin, Gentran 40, gelatin, hydroxyethylamyle etc.

Phrase " pharmaceutically acceptable " refers to that molecular entity and composition have physiology admissibility and after being applied to people, usually can not producing irritated or similar reaction, as having a stomach upset, dizzy etc.And by federal or state government's approved by management, or American Pharmacopeia or other generally acknowledge for animal, more specifically to what list in the pharmacopeia of people.Carrier " refer to thinner, adjuvant, vehicle or carrier that medicament is ratified.This type of pharmaceutical carrier can be sterile liquid, and the salt brine solution of such as water and oil comprise oil, animal oil, vegetables oil or synthetic oil, as peanut oil, soybean oil, mineral oil, sesame wet goods.When pharmaceutical composition intravenously administrable, salt brine solution is preferred carrier.Salt brine solution and dextrose hydrate and glycerine solution also can be used as liquid vehicle, particularly injection liquid.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skim-milk, glycerine, propylene glycol, ethylene glycol, water, ethanol etc.If needed, these compositions also comprise indivisible wetting agent, emulsifying agent and/or pH buffer reagent.

These compositions can adopt following form: solution, suspensoid, emulsion, tablet, pill, capsule, pulvis, extended release preparation, cachet, lozenge, lozenge, dispersion agent, suppository, ointment, cataplasma (paste), paste, dressing, creme, plaster, patch, aerosol, gelifying agent.Liquid dosage form is suitable for parenteral and is applied to patient, and sterile solids (as crystal or amorphous solid) is reconstructed into liquid dosage form to be suitable for parenterai administration patient.Such composition should comprise the described compound (form of preferred purifying) for the treatment of significant quantity and appropriate carrier, to provide suitable form of medication to patient.

The pharmaceutical composition being suitable for oral administration can be capsule, tablet, pulvis, granule, solution, syrup, suspension (at non-water or waterborne liquid) or emulsion.Tablet or hard gelatin capsule can comprise lactose, starch or derivatives thereof, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate, stearic acid or its salt.Soft gelatin capsule can comprise vegetables oil, wax, fat, semisolid or liquid polyol.Solution and syrup can comprise water, polyvalent alcohol and sugar.This active agents of active agents useful delay being designed for oral administration GI disintegration and/or absorption thing dressing or mix with it.Therefore, promoting agent sustained release multiple hours can be realized, and if need, can by promoting agent protection to prevent from degrading under one's belt.Preparation is used for the oral pharmaceutical composition given, and is convenient to promoting agent and discharges at specific gastrointestinal location.Orally administered pharmaceutical composition can be prepared to help promoting agent in the release of specific gastrointestinal location (due to special pH or enzyme condition).

The preparation being applicable to transdermal administration can be discontinuous patch, keeps close contact with long-time and patient's epidermis.

Be applicable to can comprising solid-state carrier as can quickly through the pulvis of nasal administration to the pharmaceutical composition of nasal cavity and lung medication.Pharmaceutical composition for nasal administration can comprise liquid vehicle, such as nasal spray or nasal drop.In addition, by deeply having a suck of gas or installing blowpipe to realize directly sucking lung.These compositions can comprise water or the oil solution of activeconstituents.Can be contained in equipment supporting specially for the composition by inhalation, include but not limited to pressurised aerosol, atomizer or insufflator, such device can be manufactured, to provide the activeconstituents of predetermined dose.

The pharmaceutical composition being suitable for administered parenterally comprises water-based and non-aqueous sterile injection solution or suspension, the solute that described solution or suspensoid can contain antioxidant, buffer reagent, fungistat and make described composition become isotonic substantially with the blood of intended recipient.Other compositions that can exist in this based composition comprise, such as water, alcohol, polyvalent alcohol, glycerine and vegetables oil.The composition being suitable for administered parenterally may reside in unitary dose or multi-dose container, the ampoule such as sealed and bottle, and under lyophilize (freeze-drying) condition can be stored in.For this situation, only needing before use to add sterile liquid carrier rapidly.Extemporaneous injection solutions and suspension can be prepared by sterilized powder, particle and tablet.Can be used for providing the suitable carrier of parenteral dosage forms of the present invention to be well-known to those skilled in the art.Example comprises: USP water for injection; Aqueous carrier, such as sodium chloride injection, ringer's inj, glucose injection, dextrose & sodium chloride injection and lactated Ringer's injection liquid; The mixable vehicle of water such as ethanol, polyoxyethylene glycol and polypropylene glycol; And non-aqueous carrier, as Semen Maydis oil, Oleum Gossypii semen, peanut oil, sesame oil, ethyl oleate, Isopropyl myristate and peruscabin.

The selection of effective dose is determined by those skilled in the art according to the consideration of the known several factors of those of ordinary skill in the art.These factors comprise the particular form of inhibitor and pharmacokinetic parameter thereof as bioavailability, metabolism, transformation period etc., these factors are established in the development sequence of routine, and these programs of utilization could obtain the approval by regulation to medical compounds usually.Other factors relevant with dosage comprise, the illness that will treat or disease, or to the benefit that normal people will obtain, the body weight of patient, the approach of medication, medication is acute or chronic, concomitant dosing, and impact give other factors well known of medicament efficiency.Therefore accurate dosage should be determined according to the particular case of the judgement of doctor and every patient.

In some embodiments, use antipyretic and/or antihistaminic agent (paracetamol and Vena) to patient, any possible DMSO infusion toxicity that freezing element contained when treating with hES-MSC to reduce is relevant.

5.12T-MSC conditioned medium and derivative

T-MSC disclosed herein can be used for producing immunosuppressant conditioned medium, and namely this substratum comprises one or more biomolecules by stem cell secretion or excretion.The immunocyte of described stem cell to one or more types multiple has detectable immunosuppression capability.In various embodiments, described conditioned medium comprises the substratum that T-MSC grown at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or more number of days.In other embodiments, described conditioned medium comprise T-MSC grown into degree of converging at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or reach 100% substratum.This conditioned medium can be used for the cultivation supporting single T-MSC group, or the cultivation of the stem cell of other type.In another embodiment, described conditioned medium comprises the substratum of T-MSC differentiation and maturation cell.In another embodiment, conditioned medium of the present invention comprises the substratum cultivating T-MSC and non-T-MSC.

Therefore, in one embodiment, the invention provides a kind of composition comprising substratum, cell lysate and/or other T-MSC derivative, wherein said T-MSC (a) sticks in matrix; B () expresses CD73, CD105, CD90, CD29, CD44, CD146, IL-10, TGFb2, HGF, but do not express IL-6, TNF α, IL-12 and/or RAGE.In another particular embodiment of the invention, said composition comprises antiproliferative reagent, as anti-MIP-1 α or anti-MIP-1 β antibody.

Thering is provided a kind of herein uses T-MSC described herein to increase as feeder cell the method for marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord hemopoietic stem cell.In some embodiments, the T-MSC being suitable for disclosure method expresses Stro-3, Stro-1, DL1 and/or Nestin.Described T-MSC also can by Section of 5.5 disclosed method modification or transformation herein to express high-caliber Stro-3, Stro-1, DL1, Nestin and/or Frizzle.In some embodiments, described T-MSC is mescenchymal stem cell.The coculture of T-MSC as described herein and marrow hemopoietic stem cells is provided herein.The coculture of T-MSC as described herein and umbilical cord hemopoietic stem cell is provided herein.

5.13 matrix comprising T-MSC and/or T-MSC derived cell system

The present invention also comprises matrix, hydrogel, support and similar comprises T-MSC and/or T-MSC-DL.T-MSC and/or T-MSC-DL can be seeded in natural substrates, as biomaterial.In some embodiments, the described support of acquisition is printed by 3 dimensions.T-MSC and/or T-MSC-DL can be suspended in the hydrogel solution be applicable to as injection.Suitable hydrogel for such composition comprises self-assembling peptides, such as RAD16.In one embodiment, wrapping celliferous hydrogel solution and can allow hardening, such as in a mold, to be formed, there is the cell dispersal matrix for transplanting wherein.T-MSC and/or T-MSC-DL can cultivate in such matrix, makes cell carry out mitotic division amplification before transplantation.Described hydrogel is, such as organic polymer (natural or synthesis), and by covalency, ion or hydrogen bond crosslinks to create the crystalline network of Three-dimensional Open, its water molecules of capturing, to form gel.Hydrogel forms material and comprises polysaccharide, such as algin and salt thereof, polypeptide, poly-phosphine piperazine and polyacrylic ester, they are cross-linked by ionic linkage or block polymer, block polymer comprises such as polyethylene oxide-polyethylene glycol block multipolymer, and they are cross-linked respectively by temperature or pH value.In some embodiments, hydrogel of the present invention or matrix are biodegradable.In certain embodiments of the present invention, described preparation comprise in-situ polymerization gel (see, such as, U.S. Patent Application Publication 2002/0022676; Anseth etc., J.Control Release, 78 (1-3): 199-209 (2002); Wang etc., Biomaterials, 24 (22): 3969-80 (2003).

In some embodiments, described polymkeric substance at least can be partially dissolved in the aqueous solution such as water with electrically charged side base or its univalent ion salt, or in aqueous alcohol solutions.There is the example of the polymkeric substance of acidic pendant groups, polymkeric substance can with cationoid reaction be poly-(phosphonitrile), poly-(vinylformic acid), poly-(methacrylic acid), vinylformic acid and methacrylic acid, the poly-multipolymer of (vinyl-acetic ester) and the polymkeric substance of sulfonation, such as, as sulfonated polystyrene.Also the multipolymer with the acidic pendant groups formed with vinyl ether monomers or polymer reaction by acrylic or methacrylic acid can be used.The example of acidic-group is hydroxy-acid group, sulfonic acid group, halo (preferred fluoro) alcohol groups, phenol OH group and acid OH group.

T-MSC, T-MSC-DL and/or coculture can be seeded to three-dimensional framework or support, and are implanted in body.This framework can be transplanted together with any one or more somatomedins, cell, medicine or other component, to stimulate organization formation or otherwise strengthen or improve enforcement of the present invention.

The example of the support that can use in the present invention comprises non-woven mat, porous foam or self-assembly polypeptide.Can use processbearing astrocyte nonwoven fabric mat, the absorbable multipolymer that this fiber is synthesized by ethanol and lactic acid forms (as PGA/PLA) VICRYL, Ethicon, Inc., Somerville, N.J.).Foam also can be used as support, and this foam is by such as consisting of poly-(6-caprolactone)/poly-(oxyacetic acid) (PCL/PGA) multipolymer.This multipolymer is formed (such as, see, U.S. Patent number 6355699) by such as lyophilize or freeze-dry process.

T-MSC and/or T-MSC-DL also can be seeded in or contact physiologically acceptable stupalith, include but not limited to, single-, two-, three-, α-three-, β-three and four calcium phosphate, hydroxyapatite, fluorapatite, calcium sulfate, calcium fluorochemical, calcium oxide, calcium carbonate, magnesium calcium phosphate, bioactivity glass as and their mixture.The stupalith that multiporous biological commercially available is at present compatible comprises (CanMedica Corp., Canada), (Merck Biomaterial France, France), (Mathys, AG, Bei Telahe, Switzerland) and mineralized collagen bone collection product, such as HEALOS ^tM(DePuy, Inc., Lei Namu, Massachusetts) and rHAKOSS ^tMwith (Orthovita, Malvern, Pa.).This framework can be a mixture, blend or mixture that is natural and/or synthetic materials.

In another embodiment, T-MSC and/or T-MSC-DL can be seeded in or contact felt, and this felt can be by, as multifilament is formed.This multifilament can by the material of bio-absorbable as PGA, PLA, PCL multipolymer or blend, or hyaluronic acid is made.

In another embodiment, T-MSC and/or T-MSC-DL can be seeded in foam stand, and this support can be composite structure.This type of foam stand can be molded as useful shape, such as, and to be repaired, the part of health ad hoc structure of replacing or supplementing.In some embodiments, described framework, before inoculation cell of the present invention, uses such as 0.1M acetic acid treatment, then at polylysine, PBS and/or collagen protein incubation, to strengthen cell adhesion.The outer surface of matrix can be modified to improve the differentiation of biological cells and tissues, such as gas ions bag is by matrix, or add one or more protein (such as collagen, spandex fiber, reticulin fiber), glycoprotein, glucose glycosaminoglycan (such as heparin sulfate, chrondroitin-4-vitriol, chrondroitin-6-vitriol, dermatan sulfate, keratan sulfate etc.), extracellular matrix and/or other matrix, as but be not limited to gelatin, alginate, agar, agarose and vegetable jelly etc.

In some embodiments, described support comprises does not make its thrombosed material, or support is by this material processing.These process and material also can promote and maintain endothelial growth, migration and extrtacellular matrix deposition.The embodiment of these materials and treatment includes but not limited to natural materials such as basement membrane proteins, as ln and IV Collagen Type VI, and synthetic materials, the polyurethane-urea silicone of such as EPTFE and segmentation, such as PURSPAN ^tM(The PolymerTechnology Group, Inc., Berkeley, California).Described support can also comprise antithrombotic agent, such as heparin.Before inoculation stem cell, this support also can be processed, to change surface charge (such as, wrapping by plasma body).

T-MSC and/or T-MSC-DL of 5.14 immortalizations

Mammiferous T-MSC and/or T-MSC-DL contains any suitable carrier of GPG to realize immortalization with good conditionsi by transfection.The protein of these genes encodings promotes the Growth of Cells of transfection under proper condition, makes described growth promote that the output of protein and/or activity sound similarly being extraneous factor.In one preferred embodiment, GPG is oncogene, such as but not limited to adenovirus or the E7 albumen human papillomavirus of v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyomavirus large T antigen, E1a.

To realize under the example that growth promotion albumen regulates can pass through, be placed in by growth promoting gene in extraneous regulatable promotor, such as, the controlled activity of promotor such as, in, transfected cell temperature or change with the composition of cells contacting substratum.In one embodiment, gene expression system (reference Gossen etc., Proc.Natl.Acad.Sci.USA 89:5547-5551,1992 that tsiklomitsin (tet) can be used to control; Hoshimaru etc., Proc.Natl.Acad.Sci.USA 93:1518-1523,1996).When there is not tet, the trans-acting factor (tTA) that in carrier, tet controls activates transcribing of phCMV*-1 by force, and wherein phCMV*-1 merges the minimum human cytomegalic inclusion disease virus promotor to tet operon sequence.TTA's is the fusion rotein checking (tetrahedron) of acid domain of the tet resistance operon in colibacillary transposon-10 source and the VP16 of hsv.Tet (such as, 0.01 ~ 1.0 μ g/mL) that is low, non-toxic concn almost eliminates the trans-activation of tTA completely.

In one embodiment, carrier also comprises the gene of encoding selectable markers (such as giving the protein of drug resistance).This bacterial neomycin resistance gene (neo ^r) be exactly a kind of like this mark, this mark also may be used in the present invention.By carrying neo for method choice known to persons of ordinary skill in the art ^rcell, such as, in growth medium, add 100-200 μ g/mL G418.

Realize transfection by any one method in the various methods that those skilled in the art are known, include but not limited to retroviral infection.Usually, to the mixture incubation of cell culture transfection by being formed with conditioned medium and the DMEM/F12 that comprises N2 fill-in.Wherein, conditioned medium is from the production clone for described carrier.Such as, by containing in the mixture of the DMEM/F12 of N2 fill-in incubation at 1 times of volume conditions substratum and 2 times of volumes about 20 hours, the stem cell culture of preparation as mentioned above can be infected in vitro, such as 5 days.Then the transfectional cell of selectable markers can be carried by method choice as described above.

After transfection, culture goes down to posterity to and allows the surface of propagation, such as, allows the cell of at least 30% to double in 24 hours periods.Preferably, described matrix is poly ornithine/Laminin substrate, comprises tissue culturing plastic and scribbles poly ornithine (10 μ g/mL) and/or ln (10 μ g/mL), polylysine/Laminin substrate or the surface with fibronectin process.Then, within every 3-4 days, raise culture with growth medium, in growth medium, or can not supplement one or more propagation enhancement factors.When the degree of converging of culture is less than 50%, propagation enhancement factor can be added in growth medium.

Standard technique can be used to go down to posterity T-MSC and/or the T-MSC-DL clone of described Conditional immortalization, as when 80-95% degree of converging, use trypsin digestion and cell.In some embodiments, be conducive to maintaining selection (such as, adding G418 in containing the cell of neomycin resistance gene) up to about 20 generations.Cell can also be freezing to carry out standing storage in liquid nitrogen.

Can from the people T-MSC clone of Conditional immortalization as above separating clone clone.Usually, standard technique can be used to be separated this cloned cell line, as cloned ring and amplification by limiting dilution assay or use.Usually can to raise as described above and go down to posterity cloned cell line.

The people T-MSC clone of Conditional immortalization can be, but needs not be pure lines.Usually the production of albumen or the active and people T-MSC clone of inductive condition immortalization differentiation is promoted by Developing restraint under the culture condition promoting differentiation.Such as, if encoding growth promotes the control of the promotor that the gene of albumen is regulated by outside, condition can be changed, the composition of such as temperature or substratum, thus Developing restraint promotes transcribing of gene.For the gene expression system that tsiklomitsin discussed above controls, differentiation can be realized by adding tsiklomitsin Developing restraint transcribing of gene of promotion.Usual 1 μ g/mL tsiklomitsin process is enough to cause differentiation for 4-5 days.In order to promote further differentiation, extra medicament can be added in growth medium.

5.15 test

T-MSC and/or T-MSC-DL can be applicable to test to determine culture condition, environmental factors, molecule (such as, biomolecules, little inorganic molecule etc.) etc. the impact on stem cells hyperplasia, amplification and/or differentiation, compared with not being exposed to such condition with T-MSC and/or T-MSC-DL.

In one preferred embodiment, T-MSC and/or T-MSC-DL can be used for analyzing once cells contacting molecule, propagation, the change expanding or break up.In one embodiment, such as, the invention provides a kind of compound identifying multiple T-MSC and/or the T-MSC-DL propagation of adjustment, wherein, under the condition allowing propagation, T-MSC and/or T-MSC-DL contacts this compound, compared with wherein not contacting with this compound with T-MSC and/or T-MSC-DL, if this compound causes the detectable change of the propagation of T-MSC and/or T-MSC-DL, so this compound is accredited as the compound regulating T-MSC and/or T-MSC-DL propagation.In a concrete embodiment, described compound is identified is antiblastic.In another embodiment, described compound is identified is propagation toughener.

In another embodiment, the invention provides a kind of compound identifying multiple T-MSC and/or the T-MSC-DL amplification of adjustment, wherein, under the condition allowing amplification, T-MSC and/or T-MSC-DL contacts this compound, compared with wherein not contacting with this compound with T-MSC and/or T-MSC-DL, to increase detectable change to T-MSC and/or T-MSC-DL if this compound causes, so this compound is accredited as the compound regulating T-MSC and/or T-MSC-DL amplification.In a concrete embodiment, described compound is identified is amplification inhibitor.In another embodiment, described compound is identified is amplification toughener.

In another embodiment, the invention provides a kind of compound identifying multiple T-MSC and/or the T-MSC-DL differentiation of adjustment, wherein, under the condition allowing differentiation, T-MSC and/or T-MSC-DL contacts this compound, compared with wherein not contacting with this compound with T-MSC and/or T-MSC-DL, if this compound causes break up detectable change to T-MSC and/or T-MSC-DL, so this compound is accredited as the compound regulating T-MSC and/or T-MSC-DL differentiation.In a concrete embodiment, described compound is identified is differentiation inhibitors.In another embodiment, described compound is identified is differentiation toughener.

The treatment use of the mescenchymal stem cell of 5.16 derived from human embryonic stems

The mescenchymal stem cell (BM-MSCs) deriving from marrow has been used to the treatment of the autoimmune disorder of being correlated with based on T cell, comprise multiple sclerosis (MS), but because limited source, quality are unstable and to the worry used from adult tissue's cell biological safety, make mesenchymal stem cells MSCs be restricted as auxiliary therapeutical agent.

As herein describedly produce the novel method of mescenchymal stem cell from embryonic stem cell, and make the new T-MSC that produces in this way, provide the new therapy for the relevant autoimmune disorder, particularly multiple sclerosis of T cell,

In some embodiments, the mouse to EAE morbidity uses T-MSC, reduces the disease score of these animals significantly.Described scoring reduces along with reducing demyelination, the infiltration of T cell and the microglia reaction of central nervous system, and Immunosuppression cell proliferation and differentiation in vitro.

In some embodiments, use T-MSC to the EAE mouse after seizure of disease, also can be observed disease score and decline gradually.In some embodiments, T-MSC has this prophylactic and therapeutic effect simultaneously.

In some embodiments, the immunosuppressive activity of T-MSC causes by irradiated T-MSC to these prophylactic effects, and these by the impaired myelin of the unlikely replacement of irradiated T-MSC, but decline disease score effectively.In one embodiment, the life-span of T-MSC is not shortened in radiation.

In some embodiments, the result for the treatment of of T-MSC relates to immunosuppression, CO2 laser weld and regeneration.

In some embodiments, there is less inflammatory Tcell and less T cell infiltrates into spinal cord in their central nervous system in the EAE mouse of T-MSC process.Described T-MSC can reduce the damage and symptom that are caused by inflammatory Tcell, makes them have treatment for all autoimmune disorders relevant to T cell and prevention.T-MSC also reduces demyelination.

The result that the characteristic of T-MSC all obtains with the mescenchymal stem cell of derived from bone marrow forms sharp contrast.BM-MSCs is the propagation that stimulates in response to external low dosage anti-CD 3 antibodies of suppressor T cell only, and even strengthen Th1 and Th17 Premeabilisation of cells to central nervous system.Have been found that autoreactive effect CD4 ⁺t cell is relevant with the pathogeny of some autoimmune diseases, comprises multiple sclerosis, Crohn disease and rheumatoid arthritis.These CD4 ⁺t cell comprises Th1 and Th17 cell.People BM-MSCs only has the faint or negligible effect (.2008a such as Gordon to the treatment of EAE mouse; The .2005 such as Zhang; The .2012 such as Payne).Nearest report display, the mescenchymal stem cell treatment EAE mouse in end user's umbilical cord source, disease score is only adjusted downward to 3.0 (Liu et al.2012) from 3.5.Result herein and highlight novelty and the practicality of T-MSC of the present invention from results of these researchs.

In addition, BM-MSC and T-MSC has closely similar entirety and transcribes spectrum, but some proinflammatory and anti-inflammatory factors of differential expression.In these factors, the expression level of the IL-6 of BM-MSCs is higher than T-MSC.In addition, in vitro after IFN γ stimulates, the expression of the IL-6 of BM-MSCs raises 1 times, but the IL-6 of T-MSC expression still keeps low-level.

IL-6 is a kind of pleiotropic cytokines, relates to the crosstalk of hematopoiesis/between immunocyte and stroma cell, comprises beginning and the termination of inflammation.IL-6 can promote differentiation and the function (Dong, 2008) of Th17 cell.The monocytic IL-6 level of multiple sclerosis patient blood and cerebral tissue raises (Patanella etc., 2010), and IL-6 level also raises (Sethe et al, 2006) in the serum of the elderly.The mouse lacking IL-6 receptor alpha is opposing EAE (Leech etc., 2012) when T cell sensitization.The locus specificity IL-6 that CNS produces can reorientate and strengthen the immune response (Quintana etc., 2009) of EAE, but IL-6 neutralizing antibody can reduce the inflammatory reaction (Gijbels et, 1995) of EAE mouse.Therefore, IL-6 has become the promising therapeutic targets for the treatment of multiple sclerosis.

When MSC treats MS and EAE, the immunomodulatory of periphery T cell activity, nervous cell regenerating and neurocyte reparation are by therapeutic action (Al Jumah and Abumaree, 2012 extensively approved; Auletta etc., 2012; Morando etc., 2012).But the long-term functional neurosurgery of patient MS recovers and the lasting remission state of an illness needs to repair impaired blood brain barrier and blood spinal cord barrier (Correale and Villa, 2007; Minagar etc., 2012).In other words, MS is a kind of struvite, neurodegeneration and vascular conditions, and effective treatment needs for all three parts.

The characteristic of T-MSC makes them be specially adapted to treat the relevant autoimmune disease of T cell, especially multiple sclerosis.Especially, T-MSC can reduce the disease score of EAE mouse, but also reduces the propagation of demyelination and reduction Th1 and Th17 cell, and the low expression of IL-6.The autoimmune disorder that the T cell that latter two characteristics makes them be suitable for treating other is correlated with.In addition, T-MSC passes the ability of blood brain barrier and blood spinal cord barrier, makes them have larger advantage in the autoimmune disorder that treatment is relevant to central nervous system with other with prevention multiple sclerosis.

In one embodiment, the invention provides the method for a kind for the treatment of autoimmune disorder relevant to T cell with prevention, wherein, comprise to the step having its experimenter of needs to use medicine, described medicine is the solution of the treatment significant quantity comprising MSC, cell culture or pharmaceutical preparation.The autoimmune disease that T cell is relevant includes but not limited to multiple sclerosis, inflammatory bowel, Crohn's disease, graft versus host disease (GVH disease), systemic lupus erythematous and rheumatoid arthritis.Experimenter is Mammals, most preferably people.Described solution, cell culture or pharmaceutical preparation can comprise by radiation or the T-MSC without radiation.Described solution, cell culture or pharmaceutical preparation are preferably by drug administration by injection.

Multiple sclerosis has been divided into four hypotypes: recurrent/persistence; Secondary Progressive symmetric erythrokeratodermia; Former Progressive symmetric erythrokeratodermia; Recur with Progressive symmetric erythrokeratodermia.The feature of this recurrent/persistence hypotype is that unpredictable recurrence is succeeded by long-time alleviation.Secondary Progressive symmetric erythrokeratodermia MS individuality often starts with recurrent/persistence MS, is then do not have paracmastic Progressive symmetric erythrokeratodermia to fail.Former Progressive symmetric erythrokeratodermia MS describes the individuality never having alleviation after symptom appears in minority.Progressive symmetric erythrokeratodermia recurrence is modal hypotype, has stable neural deterioration, and suffers the feature of acute attack.

Experimenter herein for there being this to need provides a kind of method for the treatment of or prevention multiple sclerosis, wherein, the method comprises the step to there being its experimenter of needs to use medicine, and described medicine is the solution of the treatment significant quantity comprising MSC, cell culture or pharmaceutical preparation.Described multiple sclerosis can be relapsing/remitting multiple sclerosis, Progressive symmetric erythrokeratodermia/Relapsing Multiple Sclerosis disease, primary multiple sclerosis or Secondary cases multiple sclerosis.Described solution, cell culture or pharmaceutical preparation can comprise by radiation or the T-MSC without radiation.Described solution, cell culture or pharmaceutical preparation are preferably by drug administration by injection.

The series of symptoms occurred in multiple sclerosis comprises four limbs sensory disturbance, optic nerve function obstacle, pyramidal tract dysfunction, vesical dysfunction, intestinal dysfunction, sexual dysfunction, ataxia and diplopia and attacks.

Another embodiment of the invention is a kind of method for the treatment of multiple sclerosis, wherein, comprises the step to there being its experimenter of needs to use medicine, and described medicine is the solution of the treatment significant quantity comprising MSC, cell culture or pharmaceutical preparation.Wherein detectable improvement has at least one symptom, at least two kinds of symptoms, at least three kinds of symptoms, at least four kinds of symptoms, at least five kinds of symptoms or all these symptoms.

Expanded disability status scale (EDSS) is the judgement criteria that the most frequently used evaluation suffers from multiple sclerosis clinical state.It is disabled according to multiple independent parameter measurement: intensity, sensation, brainstem function (language and swallow), coordination, vision, cognition and intestines/bladder incontinence.It is a generally acknowledged disabled meter of MS.And performing gets up is not difficult or consuming time especially.This EDSS quantizes eight disabled function systems (FS) and each function system allowing neurologist to distribute in these marks (Kurtzke 1983).

The following defined function system of Kurtzke:

Cone-shaped

Cerebellum

Brain stem

Sensation

Intestines and bladder

Vision

Brain

Other

EDSS scoring 1.0 to 4.5 refers to complete ambulant multiple sclerosis patients.EDSS is by being damaged to walking definition 5.0 to 9.5.The clinical meaning of each possible outcome is as follows:

0.0: neurologic examination is normal;

1.0: without disabled, minimum abnormal sign in a function system;

1.5: without disabled, minimum abnormal sign in 2/7 function system;

2.0:1/7 there is lowest capability to lose in function system;

Lowest capability is had to lose in 2.5: two function systems;

Moderate Disability is had in 3.0: one function systems; Or have slight Disability, although can walk about completely in three or four function systems;

3.5: can walk about completely, but there are moderate Disability and 1 or 2 slight Disability of function system in a function system, or 2 function system moderate Disabilities, or 5 slight Disabilities of function system;

4.0: although there is the Disability of relative severe, without assistance, can walk about completely, support oneself, reach about 12h/ days; Under not having help or nonstop situation, can walk about 500m;

4.5: without assistance, in one day, about most of the time can walk about completely, can work a whole day, may limit a bit or need basic help by Shou ー in abundant activity in addition; With the Disability of relative severe for feature; Under not having help or nonstop situation, can walk about 300m;

5.0: do not want help and can walk about 200 meters.The all daily routines of disabled obstruction;

5.5: 100 meters can be walked about, all daily routines of disabled obstruction;

6.0: no matter whether have a rest, need intermittence or one-sided lasting assistance (walking stick, crutch or support) to walk 100 meters;

6.5: need the bilateral support (walking stick, crutch or support) continued to walk 20 meters with not resting place;

7.0: even if can not walk more than 5 meters when helpful, be substantially limited in wheelchair, oneself operational standard wheelchair is walked about; Within one day, treat in wheelchair for up to about 12h;

7.5: a few step can not be walked more, be limited in bed, may want help during transfer.Can to control oneself upper wheelchair, but electrically propelled chair may be needed to complete all daily routines;

8.0: be substantially limited in bed, chair or wheelchair, but can not lie in bed;

8.5: one mosts of the time were substantially limited in bed, effectively can use arm, remained some self-care abilities;

9.0: the patient that bed is helpless, can exchange and diet;

9.5: effectively can not to exchange or diet/to swallow;

10; Dead because of MS.

There is provided a kind of method being used for the treatment of the multiple sclerosis of its experimenter of needs herein, the method comprises the step to there being its experimenter of needs to use medicine, and described medicine is the solution of the treatment significant quantity comprising T-MSC, cell culture or pharmaceutical preparation.Wherein, the scoring of described experimenter's Expanded disability status scale (EDSS) at least improves one point, and preferably at least improves 2 points.

Also have other to be used for the treatment of and to have prevented the medicine of polyvoltinism sclerosis, include but not limited to FTY720, thyroliberin (ACTH), methyl meticortelone, dexamethasone, IFN β-1a, IFN-1b, acetic acid copaxone, endoxan, methotrexate, azathioprine, CldAdo, S-Neoral, mitoxantrone and sulfasalazine.

Therefore, method of the present invention also can comprise further and gives one or more other healing potions to experimenter, and described medicament includes but not limited to FTY720, thyroliberin (ACTH), methyl meticortelone, dexamethasone, IFN β-1a, IFN-1b, acetic acid copaxone, endoxan, methotrexate, azathioprine, CldAdo, S-Neoral, mitoxantrone and sulfasalazine.In another embodiment, other healing potion described can be used after T-MSC uses again, or and T-MSC use simultaneously, or can conjugation or be connected to T-MSC.

Except T cell, T-MSC also has the powerful immune suppression function of B cell, dendritic cell, neutrophilic granulocyte, NK cell, scavenger cell and other struvite and immune correlation function.Therefore, with T cell, B cell, struvite and/or that congenital immunity is relevant autoimmune disorder can be treated with disclosed T-MSC, and described autoimmune disorder includes but not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmunity Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Autoimmune Inner Ear Disease, autoimmunity lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), behcets disease, bullous pemphigoid, myocardosis, celiac disease-dermatitis, chronic fatigue syndrome acquired immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, chronic obstructive pulmonary disease (COPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Podgorica is sick, dermatomyositis, JDMS, discoid lupus erythematosus, idiopathic mixed cryoglobulin mass formed by blood stasis, fibromyalgia-fibromyositis, Graves disease, Guillain-Barre&1& syndrome, struma lymphomatosa, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgANP, insulin-dependent diabetes (I type), type ii diabetes, juvenile arthritis, lupus, Meniere, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary Agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, auspicious special syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren syndrome, stiff people's syndromes, multiple takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wei Genashi granuloma or any acute or burning that chronic inflammatory diseases is relevant, surgical injury and allergy.

T-MSC can be divided into various kinds of cell system, includes but not limited to adipocyte, sarcoplast, neural lineage cell, scleroblast, inoblast, chondrocyte, mesenchymal cell.The derivative cell (T-MSC-DL) of these T-MSC can be used for the damage for the treatment of Various Tissues, and can be used for that organizational project, tissue repair, tissue regeneration, joint repair, tendon healing, reticular tissue healing, neural clan tissue and cytothesis, fatty tissue healing, knitting, skin healing, other wound healing, muscle healing, healing cartilage, unstriated muscle heal, myocardium healing, epithelium healing, Ligament healing, matrix reparation etc.

Particularly, T-MSC can be divided into neural lineage cell, is used for the treatment of many sacred diseases, includes but not limited to disease of failing in writing, Alzheimer, amyotrophic lateral sclerosis, aphasia, apraxia, arachnoiditis, ataxia telangiectasia, hyperkinetic syndrome, auditory processing obstacle, autism, alcoholism, A Si Burger syndromes, bipolar affective disorder, bell's palsy, brachia plexus injury, brain injury, brain injury, cerebral tumor, canavan's disease, Kapp lattice draw syndromes, cusalgia, central pain syndrome, central pontine myelinolysis, central nucleus myopathy, head deficiency disorder, cerebral aneurysm, cerebral arteriosclerosis, encephalatrophy, cerebral gigantism, cerebral paralysis, cerebral vasculitis, Cervical Vertebral Canal Procedures is narrow, charcot marie tooth, A Qi deformity, tarantism, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, two Cotards are strangled by section, stupor, Complex regional pain syndrome, compression neuropathy becomes, facial diplegia,congenital, corticobasal degeneration, cranial arteritis, a word used in person's names seam early closes, Creutzfeldt-Jakob disease, accumulation trauma, hypercortisolism, cytomegalovirus infection (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, the gloomy disease in road, moral Morsier syndrome, Dejerine-Klumpke benumbs, this disease of Dejerine-Sarasota, delayed sleep phase syndrome, senile dementia, dermatomyositis, grow dyskinesia, diabetic neuropathy, diffuse sclerosis, Down's syndrome, de Morsier's syndrome, vegetative dystonie, dyscalculia, dysgraphia, Dyslexia, myodystonia, empty sella syndrome, encephalitis, Naoning tablet, encephalotrigeminal angiomatosis, scoracratia, epilepsy, erb's palsy, erythromelalgia, essential tremor, Fabry disease, Fa Er Cotard, faint, familial spastic paraplegia, febrile convulsion, take snow syndrome, Freed ataxia, fibromyalgia, Foville's syndrome, fetal alcohol effect, gaucher's disease, grignard syndrome, giant cell arteritis, cytomegalovirus infection, globoid cell leukodystropy, gray matter heterotopia, Guillain Barre syndrome, HTLV-1 associated myelopathy, Halle Wo Deng-Si Paci disease, injury of head, headache, mimic convulsion, hereditary spastic paraplegia, heredopathia atactic polyneuritifor, auris blister epidemic disease, zoster, Pingshan Mountain syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hypercortisolism, anoxic, immune mediating encephalomyelitis, inclusion body myositis, bloch-Siemens syndrome, children's's phytanic acid storage disease, children's's refsum's disease, infantile spasms, inflammatory myositis, entocranial cyst, intracranial hypertension, Zhu Baite syndrome, OK a karaoke club gram syndrome, Kearns-Sayre syndrome, sweet is that enlightening is sick, dancing eyes syndrome, Klippel Feil syndrome, Krabbe disease, Kugelberg-Welander disease, myoclonic epilepsy, Lambert-Eton myasthenic syndrome, the acquired aphasia of the epilepsy that occurs together, lateral bulbar (Warren shellfish lattice) syndromes, learning disorder, Leigh disease, lennox-Gastaut syndrome, self-mutilation syndrome, leukodystrophy, dementia with Lewy body, congenital agyria, apallic syndrome, Lu Jialeishi disease (see amyotrophic lateral sclerosis), prolapse of lumbar intervertebral disc, lumbar spinal stenosis, Lyme disease-neural system sequela, Machado-Joseph disease (3 type spinocebellar ataxia), macrencephaly, megalopia, macrencephaly, Melkersson Rosenthal syndrome, Meniere, meningitis, Menkes disease, different leukodystrophy, microcephalus, micropsia, migraine, Miller takes snow syndrome, transient apoplexy (transient ischemic attack), acousticophobia, mitochondrial encephalomyopathy, Mobius syndromes, single limb myatrophy, motor neuron, motor skill disorder, moyamoya, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis), myasthenia gravis, myelinoclasis diffuse sclerosis, myoclonic encephalopathy of infant, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neurofibromatosis, neuroleptic malignant syndrome, the neurological manifestations of AIDS, the neurological sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neurone displacement is abnormal, Niemann-Pick disease, non-24-hour sleep-wake syndrome, Nonverbal learning disabilities, Ao Shaliwen-macLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, the former syndrome in land for growing field crops, olivopontocerebellar atrophy, heterphoria epidemic disease contraction syndrome, optic neuritis, orthostatic hypotension, excessive use is comprehensive, palinopsia, paresthesia, Parkinson's disease, myotonia congenita, paraneoplastic disease, paroxysmal is shown effect, parry-Romberg syndrome, Pelizaeus Merzbacher disease, periodic paralysis, peripheral neuropathy, pervasive developmental disorders, opticity sneezing reflex, refsum disease, Pick's disease, pinch nerve, pituitary tumor, PMG, polyneuropathy, poliomyelitis, polymicrogyria, polymyositis, postpoliomyelitis syndrome, post-herpetic neuralgia (PHN), postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy, progressive multifocal leukoencephalopathy, stein-leventhal syndrome, pseudotumor cerebri, tetraplegia, rabies, Ramsey Hunt's syndrome I type, Ramsey-Hunt's syndrome II type, Ramsey-Hunt's syndrome type III (see Ramsey-Hunt's syndrome), Lars nurse encephalitis, reflex neurovascular is malnutritive, refsum's disease, repetitive pressure damages, restless leg syndrome, retrovirus associated myelopathy, the special syndrome of thunder, Reye syndrome, rhythmic exercise obstacle, Lei Er syndrome, Saint Vitus dance, sandhoff disease, schilder, Schizencephaly, sensory integrative dysfunction, every-hypoplasia of optic nerve, shaken baby syndrome, zoster, shy-Drager syndrome, sjogren syndrome, sleep apnea, sleeping sickness, be satiated with food (Snatiation), sotos' syndrome, spasm, spina bifida, Spinal injury, tumor of spinal cord, Duchenne-Arandisease, spinocebellar ataxia, fissure, Si Dier-Jason Richardson-Ou Sizusiji is sick, stiff man syndrome, apoplexy, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, surface siderosis, Sydenham chorea disease, faint, Synesthesia, syringomyelia, tarsal tunnel syndrome, tardive dyskinesia, Delayed onset mental disorder, Tarlov tumour, Tay, temporal arteritis, tetanus, tethered cord syndrome, myotonic cataract, syndrome of chest outlet, trigeminal neuralgia, Tuo Deshi paralyses, Tourette Syndrome, toxic encephalopthy, transient ischemic attack, Transmissible spongiform encephalopathy, transection myelitis, cerebral trauma, tremble, trigeminal neuralgia, tropical spastic paraplegia, trypanosomiasis, epiloia, Ubisiosis, unipolar depression, Von Hippel-Lindau disease (VHL), this section's encephalomyelitis (VE) of prestige stream, fort, Wahlen Cotard, Wo Nike-Huffman disease, west's syndrome, whiplash injury, williams syndrome, Wilson's disease.

5.17 use T-MSC as delivery system

Because T-MSC of the present invention has shown the ability through blood brain barrier and blood-spinal cord barrier uniqueness, another embodiment of the invention is that a kind of T-MSC of use passes blood brain barrier and/or blood-spinal cord barrier to send the method for medicament, the medicament of the method is connected with T-MSC or puts together to form mixture, and T-MSC – agent complexes is administered to experimenter, wherein T-MSC through blood brain barrier and/or blood-spinal cord barrier to send medicament to central nervous system.T-MSC can be unicellular, cell culture, solution or pharmaceutical preparation form.Medicament will include but not limited to chemical, medicine, protein, DNA, RNA, antibody and small molecules.

Another embodiment of the invention be a kind of through blood brain barrier and/or blood-spinal cord barrier to send the delivery system of medicament, this system comprises medicament and puts together with T-MSC or be connected.

The described ability through blood brain barrier and blood-spinal cord barrier will can be used for treatment and preventing disease, include but not limited to multiple sclerosis, cancer, Parkinson's disease, Alzheimer, Huntington's disease, meningitis, encephalitis, rabies, epilepsy, dementia, Lyme disease, apoplexy, amyotrophic lateral sclerosis, brain and Spinal injury.Therefore, have and need its experimenter to suffer from or riskyly to suffer from the disease relating to blood brain barrier and blood-spinal cord barrier.Therefore, another embodiment of the invention is a kind of method of disease therapy or damage, the medicament of described method is puted together or is connected to T-MSC to form mixture, and need its experimenter to use T-MSC-agent complexes to having, wherein, T-MSC is through blood brain barrier and/or blood-spinal cord barrier and by drug delivery to central nervous system, described medicament is used for the treatment of or prevents the i or I of experimenter.Because T-MSC has powerful transfer ability and penetrating power, it also can be used as carrier to transport antitumor drug and albumen to treat lesion/cancer disease.Described T-MSC can be unicellular, cell culture, solution or pharmaceutical preparation form.Medicine includes but not limited to chemical, medicine, albumen, DNA, RNA, micro--RNA, non-coding RNA, antibody, small molecules and/nano molecular.

Be used for the treatment of and include but not limited to microbiotic, antiviral agent, anti-mycotic agent, steroid, chemotherapeutic, anti-inflammatory agent, cytokine and/or synthetic peptide with prophylactic useful agents.

The proteins and peptides puted together with T-MSC will be also useful T-MSC, described proteins and peptides comprises erythropoietin (EPO), anti-beta amyloid peptide, tissue plasminogen activator (TPA), granulocyte colony-stimulating factor (G-CSF), Interferon, rabbit (IFN), somatomedin/hormone, anti-vascular endothelial growth factor peptide, anti-tumor necrosis factor peptide, nerve growth factor, pHGF, IL-2, CX3CL1, GCV, CPT-11, Isocytosine deaminase, HSV-TK, Procaine esterase, oncolytic virus, TSP-1, TRAIL, FASL, IL-10 and TGFb.Be incorporated into special acceptor and close the proteins and peptides of these acceptors, will being also useful and being that plan of the present invention connects T-MSCs.

DNA and RNA of encoding therapeutic proteins and polypeptide also can put together formation conjugate with T-MSC, to send through blood brain barrier and/or blood-spinal cord barrier.

Term " antibody " comprises polyclonal antibody, monoclonal antibody, humanization or chimeric antibody, Single chain Fv antibody fragments, Fab fragment and F (ab') ₂fragment.Polyclonal antibody is for the allos group antibody of the specific antigen epi-position comprised in antigen, and monoclonal antibody pointer is to the homogeneity group antibody of defined epitope contained by antigen.Monoclonal antibody is useful especially in the present invention.

Effectively stop disease signal path Middle molecule or acceptor molecule activation, to express and/or any medicament of effect can be used for connecting or puting together T-MSC, described disease reference and any disease through blood brain barrier and/or blood-spinal cord barrier.This medicament includes but not limited to chemical, phytochemical, medicine, biotechnological formulation, organic molecule, antibody, nucleic acid, peptide class and protein.

Also can use " bait " molecules in inhibiting signal path, be somebody's turn to do the region that " bait " molecular simulation target molecule combines and activates in path.The molecule of activation in conjunction with the bait replacing target, and can not be able to activate.

The binding domain that " dominant interference " molecule or activated molecule (activated molecule shorten, and lack activation domain) can also be used to retain suppresses.These molecules bind receptor in signal path, but not fertile, and stop receptors bind to described activated molecule.This bait molecule and dominant disturbing molecule manufacture by the method known in the art, and by connecting or being conjugated to T-MSC to send through blood brain barrier or blood-spinal cord barrier.

Through blood brain barrier and/or blood-spinal cord barrier to send the method for medicament also for diagnostic reagent, include but not limited to chemical, antibody, polypeptide, albumen, DNA, RNA.This medicament in order to be applied to diagnosis must have and is visualized and/or quantitative method.This method includes but not limited to fluorescence, biomarker, dyestuff, labelled with radioisotope and/or nano particle.

Such delivering method and delivery system will be conducive to diagnosis neurological disorder, multiple sclerosis, cancer, Parkinson's disease, Alzheimer, Huntington's disease, meningitis, encephalitis, rabies, epilepsy, dementia, Lyme disease, apoplexy, amyotrophic lateral sclerosis and brain and Spinal injury.Therefore, another embodiment of the invention is a kind of method diagnosing the illness or damage, and the method is by connecting medicament or being conjugated to T-MSC to form mixture; And T-MSC-agent complexes is applied to doubtful ill experimenter, and wherein, described T-MSC is through blood brain barrier and/or blood-spinal cord barrier and by described drug delivery to central nervous system.Described T-MSC can be unicellular, cell culture, solution or pharmaceutical preparation form.Medicament includes but not limited to chemical, medicine, albumen, DNA, RNA, antibody and small molecules.

No matter what type and the reagent whether being used for the treatment of, preventing, diagnose, put together by any method known in the art or be connected to T-MSC, including but not limited to synthetic cell epimatrix, alginic acid-poly-L-Lysine capsule and/or container.

In some embodiments, the mass production levels that industry manufactures is included in the present invention, and such method is well known in the art.In some embodiments, scale operation comprises use Hyper-STACK2D culture systems and/or microcarrier 3D bio-reactor.

6. embodiment

Embodiment 1.T-MSC's is derivative

materials and methods

Following reagent and material obtain from the following stated manufacturer:

The mTeSR1 substratum of customization: stem cells technology company limited

BMP4:Stemgent or other suppliers

SB431542:Cayman Chemical or other suppliers

A83-01:Stemgent or other suppliers

ALK5 inhibitor: Stemgent or other suppliers

Life Technologies, Inc. of the DMEM/F12:GIBCO U.S.

Life Technologies, Inc. of the alpha-MEM:GIBCO U.S.

Foetal calf serum: Life Technologies, Inc. of the GIBCO U.S. or other suppliers

HESC clone CT2 is from University of Connecticut stem cell center, cultivated for 2 generations under being made the condition of feeder cell by the mouse embryo fibroblasts of radiation (MEF), then described hESC is seeded in bag and is also cultivated (Ludwig etc. in mTeSR1 substratum by the culture dish of matrigel, 2006) (Stemcell Technologies Inc. (CA), Vancouver, Canada).Human embryo stem cell ESI-017, ESI-05, ESI-053, ESI-049 and ESI-35 buy (California) from zoic age company.

t-MSC's is derivative

As shown in Figure 1, the hESCs of about 80% is reached by 1mg/ml dispase digestion 5-10 minute by matrigel bag by degree of converging on culture dish.Then this cell cleans 1 time through mTESR1 substratum and wraps by the culture dish of matrigel with fritter or unicellular being seeded in and cultivating 12 hours in mTESR1 substratum.Then substratum is replaced by nurse cell and forms substratum, and this substratum comprises BMP4 (2-100ng/ml), or optional A83-01 (0.1-1 μM).After cultivation 48-72 hour, described cell becomes nurse cell form from hESC sample form, the feature of nurse cell form be flat, cell size increases, core/cytosol ratio is little and diffusivity cell boundaries.Described cell is digested by Tryp-LE and uses MSC growth medium to clean, and MSC grown cultures is the α-MEM comprising 20%FBS and non-essential amino acid.Then this cell is with 5000 cells/cm ²density be seeded in bag by the culture dish of matrigel.Replaced medium after 24 hours, then every 3-4 days replaced medium.After 6 days, this cytodifferentiation became spindle like cell, the form of similar typical MSCs.The form of the 2nd day nurse cell as shown in Figure 2 A.Before 5th day, the form of-T-MSC as shown in Figure 2 B.The form of T-MSC as shown in Figure 2 C.

hB-MSC's is derivative

HESC clone CT2 is divided into EB cell, then by former described method enrichment HB (Lu et etc., 2008).Reached the hEC cell Dispase (1mg/ml of 50-80% by degree of converging on matrigel culture dish at bag, 5 to 10 minutes) digestion, then following substratum is used to clean: EB basis of formation substratum or HPGM (Lonza, Walksville, Maryland) or STEMLINE I/II hematopoietic stem cell expansion substratum (Sigma, St.Louis, Missouri) or StemSpan H3000 (Stem Cell Technologies, Vancouver, Canada) or the IMDM containing 10%FBS or the DMEMM/F12 containing 10%FBS.Then cell cultures is formed in substratum at EB, this culture medium supplemented 50ng/ml VEGF (Peprotech) and 50ng/ml of BMP4 (Stemgent).Cultivate 48 hours in ultralow culture dish with the density of about 2-3 1,000,000 cells/ml.After 48 hours, the substratum of half is replaced with the fresh EB that with the addition of 25-50ng/ml bFGF and forms substratum.

After 4 days, collect the EB cell that formed in the medium and use C rypLE to digest 2-3 minute at 37 DEG C, EB cell dispersal is become unicellular.Cell is cleaned and is resuspended in EB basis of formation substratum with the density of 1-5 1,000,000 cells/ml.Described single cell suspension is with the ratio of 1:10 and angioblast growth medium (Stem CellTechnologies, Vancouver, Canada) mixing.

Being formulated as follows of parent cell growth medium: be that in the methylcellulose gum CFU substratum of the serum-free of 100ml, interpolation is as follows in total amount, VEGF, TPO, FLT3 to concentration 50ng/ml, bFGF to concentration 20-50ng/ml, 1ml EX-CYTE grows and strengthens medium supplement and 1ml Pen/Strap, mix.

Mixture is seeded in ultralow culture dish and at 37 DEG C, CO through 16G pin after vortex ₂5-9 days is cultivated in environment.

Unicellular resuspended in MSC substratum, this substratum comprises: 1) containing the α-MEM (Invitrogen) of 10-20%FBS, or 2) containing the α-MEM of 10-20%KOSR, 3) containing the DMEM in high glucose of 10-20%FBS, or 4) containing the DMEM in high glucose of 10-20%KOSR.Cell is with 100-5000 cell/cm ²density cultivate bag by the culture dish of matrigel, gelatin, vitronectin, ln, fibronectin or collagen I.Change this substratum after 24 hours and upgrade for every 2-4 days.After 6-12 days, described cell is divided into gradually and spins Duo shape cell, the similar typical MSC of form.

MSC is derived by using SB431542

This method was previously delivered (Chen etc., 2012).

result

Have been found that the method for described generation T-MSC has high-level efficiency, high yield and highly purified feature.As shown at the bottom of figure 1, at the 10th day, T-MSC has produced the MSC of >90% purity and cell count improves 10 times, and other method or do not produce any MSC, or the MSCs purity produced is very low.At the 20th day, T-MSC has produced the MSC of >99% purity and cell quantity has increased 300 times, and other method at most only increases 20 times.At the 30th day, 100,000 hESC 69{10143/003371-WO0/01055509.1} produced 50,000,000,000 T-MSC, and that is, hESC has originally increased 500000 times, but other method at most only increases 3000 times.

The sign of embodiment 2.T-MSC

The T-MSC that further employing flow cytometry immuning fluorescent dyeing analysis obtains from embodiment 1.

materials and methods

Use the feature of flow cytometry T-MSC.The PBS of the BSA containing 2% is used to clean and closing cell, and use antibody to dye to various cell surface marker according to manufacturers instruction, describedly be marked with Trop-2 (Trp-2, eBioscience), CD31, CD34, CD29, CD73, CD90, CD105, CD44, CD45, CD146, CD166, HLA-ABC HLA-DR, HLA-G (BD Bioscience or eBioscience).In nFACS LSRII flow cytometer, use FACSDiva software (BD Bioscience) to collect data.FlowJo software (Treestar) is used to analyze the data of collecting.

result

Before the adherent nurse cell obtained for 2nd day, the 5th day adherent ,-T-MSC and a 9th day adherent T-MSC carries out CD73 and Tro-2 dyeing.Described nurse cell only expresses high-caliber Tro-2 (being greater than 95%), but expresses the CD73 (Fig. 3 A) being less than 1%; At the 5th day, before described ,-T-MSC had the cell more than 50% to express Trop-2 and CD73 simultaneously, and the cell of 40% only expresses CD73 (Fig. 3 B); HESC break up the 9th day obtain T-MSC have be less than 1% cell expressing Trop-2 and the cell of 99% only expresses CD73 (Fig. 3 C).

Multiple cell surface markers of further employing flow cytometry staining analysis T-MSC, <3% cell expressing Trop-2 in result display T-MSC, <1% cell expressing CD31 and CD34, >99% cell expressing CD73, >95% cell expressing CD90, >90% cell expressing CD105, >99% cell expressing CD44, >80% cell expressing CD29 (Fig. 4 A-H).

Embodiment 3.T-MSC has stronger rejection ability than BM-MSC to T cell function in vitro

Compare the ability that hEs-MSCs and BM-MSCs suppresses the T cell of antigenic stimulation to be bred in vitro.

materials and methods

The cultivation of BM-MSCs

BM-MSCs derives from myelomonocyte (BMMNCs) or obtains BMMNCs from AllCells company (I meter Da) and Lonza (Basel, Switzerland).In order to derivative, thaw BMMNCs be seeded in the plastic ware of tissue culture, and substratum is for containing MEM+20%FBS.Started to occur adherent cell, and every 2-3 days feeder cells in front 4-5 days, until about 10-12 days, at this moment collecting cell with 3000-5000 cell/cm ²again spread.

The lymphocyte be separated from mouse peripheral lymph nodes is used to carry out the in vitro tests of T cell propagation.These lymphocytes are marked 10 clocks by 5 μMs of Fluoresceincarboxylic acid succinimide esters (CFSE) at 37 DEG C of temperature, follow the trail of cell proliferation by observation CFSE in the dilution of daughter cell.10000T-MSC or BM-MSC and 100000 lymphocytes are blended in a hole of 96 orifice plates, and be connected to the anti-CD 3 antibodies (0.3 of culture plate, 1 μ g/ml) and solvable anti-CD28 antibody (1 μ g/ml, eBioscience, California) stimulate this cell proliferation.Within 3rd day, collect this cell after stimulation, the FACS of then carry out dyeing anti-CD4 and anti-CD8 antibody (BD Bioscience, California).CFSE dilution makes CD4 respectively ⁺and CD8 ⁺the gate of T cell.

result

By the in vitro tests of mouse lymphocyte, find when anti-CD 3 antibodies (0.3 and 1 μ g/ml) stimulates, T-MSC suppresses mouse CD4 ⁺and CD8 ⁺the propagation of T cell, but the BM-MSC only ability inhibited (Fig. 5) when low dosage (such as 0.3 μ g/ml) anti-CD 3 antibodies stimulates

Embodiment 4.T-MSCs reduces the disease score of EAE mouse

Because proved that BM-MSCs can slow down the disease progression of multiple sclerosis disease mouse model-experimental autoimmune encephalomyelitis (EAE), the T-MSC obtained in embodiment 1 is injected into EAE mouse to determine whether they have identical effect.

materials and methods

SB431542 is used to derive MSC; The MSC deriving from this method will be called as hES-MSC (SB).This method is delivered (Chen et etc., 2012).

The induction of this EAE mouse model as mentioned previously (Stromnes and Goverman, 2006).C57BL/6 mouse is subcutaneous by injection myelin oligodendrocyte glycoprotein peptide 35-55 (MOG ^35-55), freund's adjuvant and Toxins, pertussis composition mixture, above kit is contained in (Hooke Laboratories company, MA, (Cat.#EK-0114)) in EAE induction agent box.The operating process of induction is according to the specification sheets of manufacturer and in the middle description such as Ge (2012).

BM-MSC, T-MSC or hES-MSC (SB)/mouse or PBS (excipient control) are by subcutaneous injection (i.p.), wherein, after immunity, injection in the 6th day is to induce model of falling ill in advance, and after immunity, injection in the 18th day is to induce the rear model of morbidity.Monitor the disease score of mouse every day, until 31 days.

Disease score system is as follows:

0: the sign not having disease

1: tail paralysis completely

2: partial hind is paralysed

3: hind limb paralysis completely

4: front quadriplegia;

5: dying

(Stromnes and Goverman, 2006)

result

As shown in Figure 6,6 days or fall ill in advance after injection, described T-MSC reduced the disease score of every day significantly, shows the preventive effect of T-MSC.The mouse of injection BM-MSC does not reduce disease score, and hES-MSC (SB) part reduces disease score effectively, but effect is good not as T-MSC.

Embodiment 5.T-MSC's is multilineage differentiated

materials and methods

the Osteogenesis of T-MSC, Subchondral drilling and lipogenesis

STEMPRO skeletonization and cartilage differentiation test kit (Invitrogen company, Grand Island, NY) for skeletonization and Subchondral drilling, and Hyclone company AdvanceSTEM Adipose Differentiation test kit (Thermo Scientific, Lip river root, the Utah State) for Adipose Differentiation, the explanation according to manufacturers operates.

result

As shown in Figure 7, T-MSC has all 3 clones that good potential is divided into mesoderm tissues: scleroblast, chondrocyte and adipocyte.Therefore, T-MSC can be used as cell source to be applied to tissue regeneration, organizational project and tissue repair.

Embodiment 6.T-MSC is different from hES-HB-MSC and BM-MSC

The gene expression pattern of T-MSC, hES-HB-MSC and BM-MSCs is compared by microarray analysis

materials and methods

In order to carry out microarray analysis, according to manufacturer specification operation, extract the RNA of 2-4 for hES-MSC or the 3rd generation BM-MSC with Trizol (Invitrogen, CA).End user HT-12v4 expresses the gene expression profile that BeadChip arrays (Illumina, San Diego, California) analyzes described cell.Use Genome StudioV2011.1 analytical data.The BM-MSC clone that 2 strains are originated different is used, and the hES-MSC system that 2 strains derive from H9 and MA09 is used.

result

As shown in Figure 8, the overall express spectra of some important cytokine of 3 kinds of different MS Cs, transcription factor, cell surface marker differs widely.T-MSC can play a part different in immunosuppression and tissue regeneration.

Reference

Al Jumah, M.A., and Abumaree, M.H. (2012) .The Immunomodulatory andNeuroprotective Effects of Mesenchymal Stem Cells (MSCs) in ExperimentalAutoimmune Encephalomyelitis (EAE) (mescenchymal stem cell (MSC) is at the immunomodulatory of experimental autoimmune encephalomyelitis (EAE) and neuroprotective): A Model of Multiple Sclerosis (MS) .International journal of molecular sciences 13, 9298-9331.

Anton, K., Banerjee, D., and Glod, J. (2012) .Macrophage-associated mesenchymalstem cells assume an activated, migratory, pro-inflammatory phenotype with increasedIL-6and CXCL10secretion (the scavenger cell mescenchymal stem cell of being correlated with present activation, migration, the phenotype of proinflammatory increases along with IL-6 and CXCL10 secretion) .PLoS One 7, e35036.

Auletta, J.J., Bartholomew, A.M., Maziarz, R.T., Deans, R.J., Miller, R.H., Lazarus, H.M., and Cohen, J.A. (2012) .The potential of mesenchymal stromal cells as a novelcellular therapy for multiple sclerosis (mesenchyma stromal cells is as the potentiality of the novel cell therapy for multiple sclerosis) .Immunotherapy 4,529-547.

Barberi, T., Willis, L.M., Socci, N.D., and Studer, L. (2005) .Derivation ofmultipotent mesenchymal precursors from human embryonic stem cells (the multipotency mesenchyme precursor of derived from human embryonic stem) .PLoS Med 2, e161.

Barry, F., Boynton, R.E., Liu, B., and Murphy, J.M. (2001) .Chondrogenicdifferentiation of mesenchymal stem cells from bone marrow:differentiation-dependentgene expression of matrix components (cartilage differentiation of mesenchymal stem cells MSCs: the differentiation dependent gene of matrix components is expressed) .Exp Cell Res 268,189-200.

Becher B, Durell BG, Noelle RJ (2002) Experimental autoimmune encephalitis andinflammation in the absence of interleukin-12 (experimental autoimmune encephalomyelitis when there is not interleukin 12 and inflammation) .The Journal of clinical investigation 110:493-497.

Benito-Leon, J. (2011) .Are the prevalence and incidence of multiple sclerosischanging (morbidity and the sickness rate of multiple sclerosis change)? Neuroepidemiology 36,148-149.

Brown, S.E., Tong, W., and Krebsbach, P.H. (2009) .The derivation of mesenchymalstem cells from human embryonic stem cells (derived from human embryonic stem mescenchymal stem cell) .CellsTissues Organs 189,256-260.

Chao, Y.X., He, B.P., and Tay, S.S. (mesenchymal stem cell transplantation weakens Blood Brain Barrier (BBB) permeability and neuroinflamation to (2009) .Mesenchymal stem cell transplantationattenuates blood brain barrier damage and neuroinflammation and protects dopaminergicneurons against MPTP toxicity in the substantia nigra in a model of Parkinson's disease, and in the black substance of parkinsonian model, protect dopaminergic neuron not by MPTP toxicity) .Journal of Neuroimmunology 216, 39-50.

Chaudhary P, Marracci GH, Bourdette DN (2006) Lipoic acid inhibits expression ofICAM-1and VCAM-1by CNS endothelial cells and T cell migration into the spinal cordin experimental autoimmune encephalomyelitis (Thioctic Acid makes the endotheliocyte of central nervous system and T cell move to the spinal cord of Experimental Autoimmune encephalomyelitis mouse and suppress the expression of ICAM-1 and VCAM-1) .Journal of neuroimmunology 175:87-96.

Chen, Y.S., Pelekanos, R.A., Ellis, R.L., Horne, R., Wolvetang, E.J., and Fisk, N.M. (2012) .Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cellsto Generate Mesenchymal Stem/Stromal Cells (small molecules Mesengenic induces people's induced multi-potent stem cells to produce mescenchymal stem cell/stroma cell) .Stem Cells Translational Medicine.

Chyou, S., Ekland, E.H., Carpenter, A.C., Tzeng, T.C., Tian, S., Michaud, M., Madri, J.A., and Lu, T.T. (2008) .Fibroblast-type reticular stromal cells regulate the lymph nodevasculature (fibroblast cell type pseudostructure Cell regulate lymphoglandula vascular) .J Immunol 181,3887-3896.

Connick, P., Kolappan, M., Crawley, C., Webber, D.J., Patani, R., Michell, A.W., Du, M.Q., Luan, S.L., Altmann, D.R., Thompson, A.J., et al. (2012) .Autologousmesenchymal stem cells for the treatment of secondary progressive multiple sclerosis:anopen-label phase 2a proof-of concept study (autologous mescenchymal stem cell is used for the treatment of Secondary cases Progressive symmetric erythrokeratodermia multiple sclerosis: the concept of a kind of open-label stage 2a proves research) .Lancet neurology 11, 150-156.

Correale, J., and Villa, A. (2007) .The blood-brain-barrier in multiple sclerosis:functionalroles and therapeutic targeting (blood-brain-barrier in multiple sclerosis: function and therapeutic target to) .Autoimmunity 40,148-160.

Costa, M., Dottori, M., Ng, E., Hawes, S.M., Sourris, K., Jamshidi, P., Pera, M.F., Elefanty, A.G., and Stanley, E.G. (2005) .The hESC line Envy expresses high levels ofGFP in all differentiated progeny (all differentiation progeny cells of human embryonic stem cell Envy express high-level GFP) .Nat Methods 2,259-260.

Crocker, S.J., Milner, R., Pham-Mitchell, N., and Campbell, I.L. (2006) .Cell andagonist-specific regulation of genes for matrix metalloproteinases and their tissueinhibitors by primary glial cells (metalloprotease and their tissue depressant are by the adjustment of primary neurogliocyte to cell and agonist-specific gene) .Journal of neurochemistry 98,812-823.

Cuccurullo C, Iezzi A, Fazia ML, De Cesare D, Di Francesco A, et al. (2006) Suppression of RAGE as a basis of simvastatin-dependent plaque stabilization in type 2diabetes (suppressing RAGE as the basis of the plaques stabilize of dependence Simvastatin in diabetes B) .Arteriosclerosis, thrombosis, and vascular biology 26:2716-2723.

Cunnea P, McMahon J, O'Connell E, Mashayekhi K, Fitzgerald U, et al. (2010) Gene expression analysis of the microvascular compartment in multiple sclerosis usinglaser microdissected blood vessels (using the genetic expression of laser microprobe dating vessels analysis capillary blood vessel room in multiple sclerosis) .Acta neuropathologica 119:601-615.

Dai H, Ciric B, Zhang GX, Rostami A (2012) Interleukin-10plays a crucial role insuppression of experimental autoimmune encephalomyelitis by Bowman-Birk inhibitor (interleukin-10 is played an important role in Inhibition test Autoimmune Encephalomyelitis by BBI) .Journal of neuroimmunology 245:1-7.

Dienz, O., and Rincon, M. (2009) .The effects of IL-6on CD4T cell responses (impact that IL-6 replys cd4 t cell) .Clinical immunology 130,27-33.

Djouad, F., Plence, P., Bony, C., Tropel, P., Apparailly, F., Sany, J., Noel, D., andJorgensen, C. (2003) .Immunosuppressive effect of mesenchymal stem cells favors tumorgrowth in allogeneic animals (immunosuppressive effect of mescenchymal stem cell is conducive to tumor growth allogeneic animal) .Blood 102,3837-3844.

Dong, C. (2008) .TH17cells in development:an updated view of their molecularidentity and genetic programming (developmental TH17 cell: their Molecular Identification and the more neodoxy of genetic programming) .Nat Rev Immunol 8,337-348.

Draper, J.S., Pigott, C., Thomson, J.A., and Andrews, P.W. (2002) .Surface antigensof human embryonic stem cells:changes upon differentiation in culture (surface antigen of human embryo stem cell: change according to differentiation in cultivation) .Journal of anatomy 200,249-258.

Drukker, M., Katchman, H., Katz, G., Even-Tov Friedman, S., Shezen, E., Hornstein, E., Mandelboim, O., Reisner, Y., and Benvenisty, N. (2006) .Human embryonic stem cellsand their differentiated derivatives are less susceptible to immune rejection than adult cells (human embryo stem cell is not vulnerable to immunological rejection with their differentiation derivative compared with adult) .Stem Cells 24,221-229.

Drukker, M., Katz, G., Urbach, A., Schuldiner, M., Markel, G., Itskovitz-Eldor, J., Reubinoff, B., Mandelboim, O., and Benvenisty, N. (2002) .Characterization of theexpression of MHC proteins in human embryonic stem cells (feature of the MHC protein expression of hESC) .Proceedings of the National Academy of Sciences of the United States ofAmerica 99,9864-9869.

English, K., Barry, F.P., Field-Corbett, C.P., and Mahon, B.P. (2007) .IFN-gammaand TNF alpha differentially regulate immunomodulation by murine mesenchymal stemcells (IFN-γ and TNF α is by mouse mesenchymal stem cells MSCs difference regulation and control immunomodulatory) .Immunol Lett 110,91-100.

Ge, S., Shrestha, B., Paul, D., Keating, C., Cone, R., Guglielmotti, A., and Pachter, J.S. (2012) .The CCL2synthesis inhibitor bindarit targets cells of the neurovascular unit, the andsuppresses experimental autoimmune encephalomyelitis (cell of CCL2 synthetic inhibitor bindarit target neural blood vessel unit, and Inhibition test Autoimmune Encephalomyelitis) .J Neuroinflammation 9,171.

Gijbels, K., Brocke, S., Abrams, J.S., and Steinman, L. (1995) .Administration ofneutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmuneencephalomyelitis and is associated with elevated levels of IL-6bioactivity in centralnervous system and circulation (use interleukin-6 (IL-6) neutralizing antibody weaken Experimental Autoimmune encephalomyelitis and to central nervous system and in circulating the biologically active level of IL-6 improve relevant) .Mol Med 1, 795-805.

Gordon, D., Pavlovska, G., Glover, C.P., Uney, J.B., Wraith, D., and Scolding, N.J. (2008a) .Human mesenchymal stem cells abrogate experimental allergicencephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration (human mesenchymal stem cell is eliminated experimental allergy myelencephalon and infiltrated along with a small amount of CNS after intraperitoneal injection) .Neurosci Lett 448,71-73.

Gordon, D., Pavlovska, G., Glover, C.P., Uney, J.B., Wraith, D., and Scolding, N.J. (2008b) .Human mesenchymal stem cells abrogate experimental allergicencephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration (human mesenchymal stem cell is eliminated experimental allergy myelencephalon and infiltrated along with a small amount of CNS after intraperitoneal injection) .Neuroscience letters 448,71-73.

Gordon, D., Pavlovska, G., Uney, J.B., Wraith, D.C., and Scolding, N.J. (2010) .Human mesenchymal stem cells infiltrate the spinal cord, reduce demyelination, (human mesenchymal stem cell infiltrates spinal cord to andlocalize to white matter lesions in experimental autoimmune encephalomyelitis in experimental autoimmune encephalomyelitis mouse, reduce demyelination, and navigate to white matter lesion) .J Neuropathol Exp Neurol 69, 1087-1095.

Grinnemo, K.H., Mansson, A., Dellgren, G., Klingberg, D., Wardell, E., Drvota, V., Tammik, C., Holgersson, J., Ringden, O., Sylven, C., et al. (2004) .Xenoreactivity andengraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium (human mesenchymal stem cell is transplanted to Xenoreactivity and the transplanting success of the rat heart muscle of infraction) .J ThoracCardiovasc Surg 127,1293-1300.

Hansen, W., Westendorf, A.M., and Buer, J. (2008) .Regulatory T cells as targets forimmunotherapy of autoimmunity and inflammation (regulatory T-cell is as the target of the immunotherapy of autoimmunization and inflammation) .Inflamm Allergy Drug Targets 7,217-223.

Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop, D.J., and Olson, L. (2002) .Marrow stromal cells form guiding strands in the injured spinalcord and promote recovery (marrow stromal cell is formed at impaired spinal cord and instructs chain and promote to recover) .Proceedings of the National Academy of Sciences of the United States of America 99,2199-2204.

Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J., and Keller, G. (2004) .Haemangioblast commitment is initiated in the primitive streak of the mouse embryo (blood vessel archeocyte starts sizing at the former bar of mice embryonic) .Nature 432,625-630.

Hwang, N.S., Varghese, S., Lee, H.J., Zhang, Z., Ye, Z., Bae, J., Cheng, L., andElisseeff, J. (2008) .In vivo commitment and functional tissue regeneration using humanembryonic stem cell derived mesenchymal cells (using the mesenchymal cell of derived from human embryonic stem to shape in vivo and functional tissue regeneration) .Proc Natl Acad Sci U S A 105,20641-20646.

Huss DJ, Winger RC, Cox GM, Guerau-de-Arellano M, Yang Y, et al. (2011) TGF-beta signaling via Smad4drives IL-10production in effector Th1cells and reducesT-cell trafficking in EAE (TGF-beta signal path drives to produce IL-10 and reduce T cell in EAE model by Smad4 and divides a word with a hyphen at the end of a line in effector T cell) .European journal of immunology 41:2987-2996.

Javazon, E.H., Beggs, K.J., and Flake, A.W. (2004) .Mesenchymal stem cells:paradoxes of passaging (mescenchymal stem cell: the antinomy gone down to posterity) .Exp Hematol 32,414-425.

Johnston, J., and So, T.Y. (2012) .First-line disease-modifying therapies in paediatricmultiple sclerosis:a comprehensive overview (a line disease-modifying therapy of children with multiple sclerosis: comprehensive review) .Drugs 72,1195-1211.

Karlsson, C., Emanuelsson, K., Wessberg, F., Kajic, K., Axell, M.Z., Eriksson, P.S., Lindahl, A., Hyllner, J., and Strehl, R. (2009) the .Human embryonic stem cell-derivedmesenchymal progenitors-Potential in regenerative medicine mesenchymal stem/progenitor cells of the hESC source (-at the potential of regenerative medicine) .Stem Cell Res 3,39-50.

Karussis, D., Karageorgiou, C., Vaknin-Dembinsky, A., Gowda-Kurkalli, B., Gomori, J.M., Kassis, I., Bulte, J.W., Petrou, P., Ben-Hur, T., Abramsky, O., et al. (2010) .Safetyand immunological effects of mesenchymal stem cell transplantation in patients withmultiple sclerosis and amyotrophic lateral sclerosis (patient of multiple sclerosis and amyotrophic lateral sclerosis transplants security and the immune effect of mescenchymal stem cell) .Arch Neurol 67, 1187-1194.

Klimanskaya, I., Chung, Y., Becker, S., Lu, S.J., and Lanza, R. (2006) .Humanembryonic stem cell lines derived from single blastomeres (human embryonic stem cell from single blastomere source) .Nature 444,481-485.

Kurtzke JF (1983). " Rating neurologic impairment in multiple sclerosis:anexpanded disability status scale (EDSS) " (neurologic impairment grading of multiple sclerosis: Expanded disability status scale EDSS)) .Neurology 33 (11): 1444 – 52.

Leech, M.D., Barr, T.A., Turner, D.G., Brown, S., O'Connor, R.A., Gray, D., Mellanby, R.J., and Anderton, S.M. (2012) .Cutting Edge:IL-6-Dependent Autoimmune Disease:Dendritic Cells as a Sufficient, but Transient, Source (forefront: IL-6 relies on autoimmune disorder: dendritic cell as abundance but of short duration source) .J Immunol.

Lin, G., Martins-Taylor, K., and Xu, R.H. (2010) .Human embryonic stem cellderivation, maintenance, and differentiation to trophoblast (derived from human embryonic stem, safeguard and be divided into nurse cell) .Methods in molecular biology 636,1-24.

Liu, R., Zhang, Z., Lu, Z., Borlongan, C., Pan, J., Chen, J., Qian, L., Liu, Z., Zhu, L., Zhang, J., et al. (2012) .Human Umbilical Cord Stem Cells Ameliorate ExperimentalAutoimmune Encephalomyelitis by Regulating Immunoinflammation and Remyelination (people's umbilical cord stem cells improves experimental autoimmune encephalomyelitis by immunity moderation inflammation and Remyelination) .Stem cells and development.

Liu, Y., Goldberg, A.J., Dennis, J.E., Gronowicz, G.A., and Kuhn, L.T. (2012) .One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotentstem cells on a fibrillar collagen coating (deriving mescenchymal stem cell (MSC) like cell from the human pluripotent stem cells single stage method of the coating being positioned at fibrillar collagen) .PLoS One 7, e33225.

Lu, S.J., Feng, Q., Caballero, S., Chen, Y., Moore, M.A., Grant, M.B., and Lanza, R. (2007) .Generation of functional hemangioblasts from human embryonic stem cells (producing functional angioblast from human embryo stem cell) .Nat Methods 4,501-509.

Lu, S.J., Ivanova, Y., Feng, Q., Luo, C., and Lanza, R. (2009) .Hemangioblasts fromhuman embryonic stem cells generate multilayered blood vessels with functional smoothmuscle cells (angioblast in human embryo stem cell source produces the multilayer blood vessel with the smooth muscle cell of function) .Regenerative medicine 4,37-47.

Lu, S.J., Luo, C., Holton, K., Feng, Q., Ivanova, Y., and Lanza, R. (2008) .Robustgeneration of hemangioblastic progenitors from human embryonic stem cells (effectively producing angioblastic progenitor cell from human embryo stem cell) .Regen Med 3,693-704.

Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T., Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R., Piekarczyk, M.S., et al. (2006) .Derivation of human embryonic stem cells in defined conditions (human embryo stem cell deriving under qualifications) .Nat Biotechnol 24,185-187.

Mahad, D.J., and Ransohoff, R.M. (2003) .The role of MCP-1 (CCL2) and CCR2in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) (MCP-1 (CCL2) and CCR2 is in the effect of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE)) .SeminImmunol 15,23-32.

McFarland, H.F., and Martin, R. (2007) .Multiple sclerosis:a complicated picture ofautoimmunity (multiple sclerosis: autoimmune complicated picture) .Nat Immunol 8,913-919.

Menge, T., Zhao, Y., Zhao, J., Wataha, K., Gerber, M., Zhang, J., Letourneau, P., Redell, J., Shen, L., Wang, J., et al. (2012) .Mesenchymal Stem Cells RegulateBlood-Brain Barrier Integrity Through TIMP3Release After Traumatic Brain Injury is (after traumatic brain injury, mescenchymal stem cell is by TIMP3 release regulation hemato encephalic barrier) .Science translationalmedicine 4,161ra150.

Minagar, A., Maghzi, A.H., McGee, J.C., and Alexander, J.S. (2012) .Emerging rolesof endothelial cells in multiple sclerosis pathophysiology and therapy (physiopathology of endotheliocyte in multiple sclerosis and the new role for the treatment of) .Neurological research 34,738-745.

Mohyeddin Bonab, M., Yazdanbakhsh, S., Lotfi, J., Alimoghaddom, K., Talebian, F., Hooshmand, F., Ghavamzadeh, A., and Nikbin, B. (2007) .Does mesenchymal stem celltherapy help multiple sclerosis patients? can (mescenchymal stem cell treatment help multiple sclerosis patients to Report of a pilot study? pilot study is reported) .Iranian journal of immunology:IJI 4,50-57.

Moore, C.S., Milner, R., Nishiyama, A., Frausto, R.F., Serwanski, D.R., Pagarigan, R.R., Whitton, J.L., Miller, R.H., and Crocker, S.J. (the astroglia cell tissue inhibiting of metalloprotease-1 (TIMP-1) promotes oligodendrocyte differentiation to (2011) .Astrocytic tissue inhibitor ofmetalloproteinase-1 (TIMP-1) promotes oligodendrocyte differentiation and enhancesCNS myelination, improve the myelin of central nervous system.).The Journal of neuroscience:the officialjournal of the Society for Neuroscience 31,6247-6254.

Morando, S., Vigo, T., Esposito, M., Casazza, S., Novi, G., Principato, M.C., Furlan, R., and Uccelli, A. (2012) .The therapeutic effect of mesenchymal stem celltransplantation in experimental autoimmune encephalomyelitis is mediated by peripheraland central mechanisms (result for the treatment of of periphery and central mechanism mediation mesenchymal stem cells transplantation experiments Autoimmune Encephalomyelitis) .Stem Cell Res Ther 3, 3.

Ohtaki, H., Ylostalo, J.H., Foraker, J.E., Robinson, A.P., Reger, R.L., Shioda, S., andProckop, D.J. (2008) .Stem/progenitor cells from bone marrow decrease neuronal deathin global ischemia by modulation of inflammatory/immune responses (ancestral cells of derived from bone marrow passes through to reduce inflammatory/immune response regulation neuronal death in global brain ischemia) .Proc Natl Acad Sci U S A105,14638-14643.

Olivier, E.N., Rybicki, A.C., and Bouhassira, E.E. (2006) .Differentiation of humanembryonic stem cells into bipotent mesenchymal stem cells (ES cell differentiation is two-way mescenchymal stem cell) .Stem Cells 24,1914-1922.

Patanella, A.K., Zinno, M., Quaranta, D., Nociti, V., Frisullo, G., Gainotti, G., Tonali, P.A., Batocchi, A.P., and Marra, C. (2010) .Correlations between peripheral bloodmononuclear cell production of BDNF, TNF-alpha, IL-6, the IL-10and cognitiveperformances in multiple sclerosis patients (BDNF that peripheral blood monocyte is produced, TNF-α, IL-6, the dependency of the cognitive function of IL-10 and patients with multiple sclerosis) .J Neurosci Res 88, 1106-1112.

Payne, N.L., Sun, G., McDonald, C., Layton, D., Moussa, L., Emerson-Webber, A., Veron, N., Siatskas, C., Herszfeld, D., Price, J., et al. (2012) .Distinct immunomodulatoryand migratory mechanisms underpin the therapeutic potential of human mesenchymal stemcells in autoimmune demyelination (potential of unique immunomodulatory and migration mechanism backer mescenchymal stem cell treatment autoimmunity demyelination) .Cell Transplant.

Peron, J.P., Jazedje, T., Brandao, W.N., Perin, P.M., Maluf, M., Evangelista, L.P., Halpern, S., Nisenbaum, M.G., Czeresnia, C.E., Zatz, M., et al. (2012) .Humanendometrial-derived mesenchymal stem cells suppress inflammation in the central nervoussystem of EAE mice (uterine endometrium mescenchymal stem cell suppresses the inflammation of the central nervous system of EAE mouse) .Stem Cell Rev 8,940-952.

Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999) .Multilineagepotential of adult human mesenchymal stem cells (the multidirectional potential of adult bone mesenchymal stem cells) .Science 284,143-147.

Pomper, M.G., Hammond, H., Yu, X., Ye, Z., Foss, C.A., Lin, D.D., Fox, J.J., andCheng, L. (2009) .Serial imaging of human embryonic stem-cell engraftment and teratomaformation in live mouse models (at the teratomatous continuous imaging of hESC's implant and formation in mouse model of living) .Cell Res 19,370-379.

Quintana, A., Muller, M., Frausto, R.F., Ramos, R., Getts, D.R., Sanz, E., Hofer, M.J., Krauthausen, M., King, N.J., Hidalgo, J., et al. (2009) .Site-specific production of IL-6inthe central nervous system retargets and enhances the inflammatory response inexperimental autoimmune encephalomyelitis (the locus specificity IL-6 that central nervous system produces is redirected the inflammatory reaction with Enhancement test Autoimmune Encephalomyelitis) .Journal of immunology 183, 2079-2088.

Rafei, M., Birman, E., Forner, K., and Galipeau, J. (2009a) .Allogeneicmesenchymal stem cells for treatment of experimental autoimmune encephalomyelitis (allochthonous bone mesenchymal stem cells treatment experimental autoimmune encephalomyelitis) .Mol Ther 17,1799-1803.

Rafei, M., Campeau, P.M., Aguilar-Mahecha, A., Buchanan, M., Williams, P., Birman, E., Yuan, S., Young, Y.K., Boivin, M.N., Forner, K., et al. (2009b) .Mesenchymal stromalcells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4Th17Tcells in a CC chemokine ligand 2-dependent manner (interstital stem cell is suppressed CD4Th17 cell by CC CCL2 dependency mode thus improved experimental autoimmune encephalomyelitis) .J Immunol182, 5994-6002.

Rochman, I., Paul, W.E., and Ben-Sasson, S.Z. (2005) .IL-6increases primed cellexpansion and survival (IL-6 increases amplification and the survival of contacted antigen cell) .Journal ofimmunology 174,4761-4767.

Ryan, J.M., Barry, F., Murphy, J.M., and Mahon, B.P. (2007) .Interferon-gammadoes not break, but promotes the immunosuppressive capacity of adult humanmesenchymal stem cells (gamma-interferon can not destroy but promote the immunosuppression capability of adult human mesenchymal stem cell) .Clin Exp Immunol 149,353-363.

Sanchez, L., Gutierrez-Aranda, I., Ligero, G., Rubio, R., Munoz-Lopez, M., Garcia-Perez, J.L., Ramos, V., Real, P.J., Bueno, C., Rodriguez, R., et al. (2011) .Enrichment of human ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capable to protect againstexperimental inflammatory bowel disease (mescenchymal stem cell that enrichment has the derived from human embryonic stem of immunosuppression and anti-inflammatory can prevent experimental inflammatory bowel diseases) .Stem cells (Dayton, Ohio) 29, 251-262.

Sethe, S., Scutt, A., and Stolzing, A. (2006) .Aging of mesenchymal stem cells (mescenchymal stem cell aging) .Ageing Res Rev 5,91-116.

Solchaga, L.A., Penick, K.J., and Welter, J.F. (2011) .Chondrogenic differentiationof bone marrow-derived mesenchymal stem cells:tips and tricks (Chondrocyte Differentiation of mesenchymal stem cells MSCs: gist and skill) .Methods in molecular biology 698,253-278.

Stromnes, I.M., and Goverman, J.M. (2006) .Active induction of experimentalallergic encephalomyelitis (activity inducement of experimental allergic encephalomyelitis) .Nat Protoc 1,1810-1819.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998) .Embryonic stem cell lines derived from humanblastocysts (human blastocyst-derived embryonic stem cell line) .Science 282,1145-1147.

Tse, W.T., Pendleton, J.D., Beyer, W.M., Egalka, M.C., and Guinan, E.C. (2003) .Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implicationsin transplantation (human bone marrow stroma stem cell suppresses allogeneic T cells propagation: the enlightenment to transplanting) .Transplantation 75,389-397.

Tyndall, A. (2011) .Successes and failures of stem cell transplantation inautoimmune diseases (transplanting the success and failure of stem cell in autoimmune disorder) .Hematology AmSoc Hematol Educ Program 2011,280-284.

Uccelli A, Prockop DJ (2010a) .Why should mesenchymal stem cells (MSCs) cureautoimmune diseases (why mesenchymal stem cells (MSCs) will treat autoimmune disorder)? CurrOpin Immunol 22:768-774.

Uccelli, A., and Prockop, D.J. (2010b) .Why should mesenchymal stem cells (MSCs) cure autoimmune diseases (why mesenchymal stem cells (MSCs) will treat autoimmune disorder)? Curr Opin Immunol, 7.

Waterman, R.S., Tomchuck, S.L., Henkle, S.L., and Betancourt, A.M. (2010) .A newmesenchymal stem cell (MSC) paradigm:polarization into a pro-inflammatory MSC1oran Immunosuppressive MSC2phenotype (new mescenchymal stem cell (MSC) example: produce a polarization as the MSC1 of proinflammatory disease or immunosuppressant MSC2 phenotype) .PLoS One 5, e10088.

Weber, M.S., Menge, T., Lehmann-Horn, K., Kronsbein, H.C., Zettl, U., Sellner, J., Hemmer, B., and Stuve, O. (2012) .Current treatment strategies for multiple sclerosis-efficacy versus neurological adverse effects (therapeutic strategy-curative effect that multiple sclerosis is current and neural system untoward reaction) .Current pharmaceutical design 18,209-219.

Wong, R.S. (2011) .Mesenchymal stem cells:angels or demons (mescenchymal stem cell: angel or demon)? J Biomed Biotechnol 2011,459510.

Xu, R.H., Chen, X., Li, D.S., Li, R., Addicks, G.C., Glennon, C., Zwaka, T.P., andThomson, J.A. (2002) .BMP4initiates human embryonic stem cell differentiation totrophoblast (BMP4 starts human embryo stem cell and is divided into trophoderm) .Nat Biotechnol 20,1261-1264.

Xu, R.H., et al.2002. " BMP4initiates human embryonic stem cell differentiation totrophoblast (BMP4 starts human embryo stem cell and is divided into trophoderm). " Nat Biotechnol 20:1261-1264.

Yamout, B., Hourani, R., Salti, H., Barada, W., El-Hajj, T., Al-Kutoubi, A., Herlopian, A., Baz, E.K., Mahfouz, R., Khalil-Hamdan, R., et al. (2010) .Bone marrowmesenchymal stem cell transplantation in patients with multiple sclerosis:a pilot study (preliminary study of Bone Marrow Mesenchymal Stem Cells Transplantation treatment multiple sclerosis) .J Neuroimmunol 227,185-189.

Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., Giunti, D., Ceravolo, A., Cazzanti, F., Frassoni, F., et al. (2005) .Mesenchymal stem cellsameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy (mescenchymal stem cell improves experimental autoimmune encephalomyelitis by inducing T cell without anergy) .Blood 106,1755-1761.

Zhang, J., Li, Y., Chen, J., Cui, Y., Lu, M., Elias, S.B., Mitchell, J.B., Hammill, L., Vanguri, P., and Chopp, M. (2005) .Human bone marrow stromal cell treatment improvesneurological functional recovery in EAE mice (human bone marrow substrate cell process improves EAE mouse Nerve functional rehabilitation) .Exp Neurol 195,16-26.

Although with reference to preferred embodiment, invention has been described, it will be understood by those skilled in the art that and can carry out various change, and available equivalents substitutes its key element, and do not exceed scope of the present invention.In addition, under the condition not departing from essential scope of the present invention and spirit, according to instruction of the present invention, many amendments can be made to adapt to specific end use, application, manufacturing condition, working conditions, composition, medium, size and/or material.Therefore, the present invention is not limited to described specific embodiment and preferred forms as described herein.This amendment will fall in the scope of claims.

The full content of all reference quoted here is that all objects are incorporated to here as a reference, is ad hoc pointed out separately as all objects are incorporated herein by reference as the full content of each publication or patent or patent application.

Claims (133)

1. produce a method for the mescenchymal stem cell (T-MSCs) of trophoblastic origin from human embryo stem cell (hESCs) or induced multi-potent stem cells (iPSCs), described method comprises:

A () cultivates the suitable first time period of hESCs or iPSCs in the substratum comprising Delicious peptide (BMP), be enough to make hESCs or iPSCs be divided into nurse cell, or together with TGFb inhibitor to improve differentiation efficiency;

B () is dispersed into nurse cell unicellular;

C described nurse cell is layered on bag by the plate of gelatin, vitronectin, ln, fibronectin, matrigel or collagen by () again; And

D () cultivates the second suitable time period of described nurse cell in mescenchymal stem cell (MSC) growth medium, wherein said growth of mesenchymal stem cells substratum contains LIF, bFGF or PDGF to improve amplification efficiency.

2. produce a method of T-MSCs from hESCs or iPSCs, described method comprises:

A () cultivates hESCs or iPSCs 2-5 days to make hESCs or iPSCs be divided into nurse cell in the substratum comprising BMP4;

B () is separated described nurse cell;

C () is from the trophoderm dispersion nurse cell of described separation;

D () is layered on bag by the culture plate of matrigel again described nurse cell; And

E () cultivates described nurse cell 4-10 days comprising in the MSC growth medium of serum, KOSR or other serum free medium, the amount of described substratum enough induces described unicellular differentiating one-tenth mescenchymal stem cell; Wherein mescenchymal stem cell described at least 90% expresses CD73.

3. method according to claim 1, wherein, described first time period is about 1 ¹/ ₂it was to about 4 days.

4. method according to claim 3, wherein, described first time period is about 2 days to about 5 days.

5. method according to claim 1, wherein, described second time period is about 4 days to about 10 days.

6. method according to claim 5, wherein, described second time period is about 6 days to about 8 days.

7. method according to claim 2, wherein, described substratum comprises TGF beta inhibitor further to improve differentiation efficiency.

8. method according to claim 7, wherein, described TGF beta inhibitor is SB431542, A83-01 or ALK5 inhibitor.

9. the method described in claim 1 and 2, wherein, before claim 1 the first step, hESCs cultivates and comprises the following steps:

I () is cultured to about 80% degree of converging on the culture plate of matrigel bag quilt;

(ii) cell dispersion under suitable conditions;

(iii) be separated; With

(iv) clean.

10. method according to claim 1, wherein, described BMP4 is BMP4, BMP2, BMP7, GDF 5 (GDP5) or its combination.

11. methods according to claim 10, wherein, the concentration of described BMP4 is about 1ng/ml to about 100ng/ml, the concentration of described BMP2 is about 100ng/ml to 500ng/ml, the concentration of described BMP7 is about 100ng/ml to about 500ng/ml, and the concentration of described GDP5 is about 10ng/ml to 50ng/ml.

12. methods according to claim 1, wherein, described BMP is being divided into the factor or peptide that functionally and are biologically equal to BMP4 in MSCs by hESCs, the derivative of BMP4, BMP4 preparation, BMP4 N-terminal peptide section, BMP4 C-terminal peptide section or its combination.

13. methods according to claim 2, wherein, the concentration of described BMP4 is about 2ng/ml to about 100ng/ml.

14. methods according to claim 2, wherein, the concentration of described BMP4 is about 1ng/ml to about 10ng/ml.

15. methods according to claim 8, wherein, the concentration of described A83-01 is about 0.1 μM to about 5 μMs.

16. methods according to claim 8, wherein, the concentration of described SB431542 is about 1 μM to about 20 μMs.

Method described in 17. claims 1 to 16, wherein, the T-MSC of described generation is CD73 ⁺, CD105 ⁺, CD90 ⁺, and purity is at least about 90%.

The MSCs (T-MSC) of 18. trophoblastic origin, it is produced by method according to claim 1 or claim 2.

19. methods according to claim 1 or claim 2, wherein, hESCs is replaced by the embryonic stem cell (ESCs) in non-human mammal source, and produces non-human mammal MSCs.

20. methods according to claim 1 or claim 2, wherein, hESCs is replaced by induced multi-potent stem cells (iPSCs), and produces the derivative T-MSCs of iPSC.

21. adipocytes, chondrocyte, scleroblast, neural lineage cell, sarcoplast, stroma cell and inoblast, it is produced by T-MSC according to claim 18.

MSCs (T-MSCs) cell mass that 22. trophoderms are derivative, it is produced by method according to claim 1.

23. cell masses according to claim 22, wherein, described T-MSCs is CD73 ⁺, CD105 ⁺, CD90 ⁺.

24. 1 kinds of methods for the treatment of experimenter's autoimmune disorder or illness, described method comprises the T-MSC that the method described in claim 1 to 17 that gives suitable dose by suitable route of administration is produced.

The method that 25. 1 kinds of subject immune regulate, described method comprises the T-MSC that the method described in claim 1 to 17 and 19 to 20 that gives suitable dose by suitable route of administration is produced.

26. 1 kinds is the methods that experimenter's prevention or Immunosuppression repel in tissue or organ transplantation process, and described method comprises the T-MSC of the production of the method described in claim 1 to 17 and 19 to 20 giving suitable dose by suitable route of administration.

27. cell masses according to claim 22, wherein, described cell is CD73 at least about 90% ⁺.

28. cell masses according to claim 22, wherein, described cell is CD73 at least about 95% ⁺.

Cell mass described in 29. claims 27 and 28, wherein, described cell is CD73 (>98%), CD90 (>95%), CD105 (>90%), CD44 (>95%), CD29 (>80%).

Cell mass described in 30. claims 27 to 29, wherein, described cell is that endothelial marker CD31 is negative and hematopietic markers CD34 is negative.

Select the mescenchymal stem cell (T-MSCs) of clinical rank trophoblastic origin to treat the method for autoimmune disorder for 31. 1 kinds, wherein, the T-MSCs of selection has following characteristics: (i) comprises cell expressing group-1 mark of >95%; (ii) cell expressing group-2 mark of >80% is comprised; (iii) cell expressing group-3 mark of <5% is comprised; (iv) IL-10 and TGF β is expressed; V () comprises the cell of expression IL-6, IL-12 and TNF α of <2%; (vi) cell of coexpression all groups-4 marks of <0.001% is comprised, wherein group-1 mark is: CD73, CD90, CD105, CD146, CD166 and CD44, group-2 mark is: CD13, CD29, CD54 and CD49E, group-3 mark is: CD45, CD34, CD31 and SSEA4, and group-4 mark is: OCT4, NANOG, TRA-1-60 and SSEA4.

32. methods according to claim 31, wherein, described cell does not express IL-6, IL-12 and TNF α.

33. methods according to claim 31, wherein, described cell does not express TGF-β 1, TGF-β 2 and IL10.

34. methods according to claim 31, wherein, described cell does not express MMP2 and RAGE.

35. methods according to claim 31, wherein, to express with IFN γ R2 with the IFN γ R1 of mesenchymal stem cells MSCs (BM-MSC) and compare, the IFN γ R1 of described T-MSC cell and IFN γ R2 expresses low.

36. 1 kinds of methods of producing the T-MSC cell mass of modification, comprise and modify mescenchymal stem cell (MSC) to produce the T-MSCs of modification, the T-MSCs of described modification has following characteristics: (i) expresses IL-10 and TGF β; And (ii) comprise the cell of expression IL-6, IL-12 and TNF α of <2%.

37. methods according to claim 36, comprise further and modify described MSC to reduce IL-6, IL-6 acceptor, IL-12, TNF α or its expression of combining.

38. methods according to claim 36, comprise further and modify described MSC to reduce the expression of MMP2, RAGE or its combination.

39. methods according to claim 36, comprise further and modify by shRNA and miRNA the expression that described MSC improves TGF-β 1, TGF-β 2 and/or IL10.

40. methods according to claim 36, the MSC comprised further described in modification reduces IFN γ R1, IFN γ R2, IFN γ or its expression level combined.

41. methods according to claim 1 or claim 2, comprise the step of T-MSC described in radiation further.

42. methods according to claim 1 or claim 2, wherein, described method produces 1 × 10 ⁶to 5 × 10 ¹⁰t-MSCs or production 1 × 10 ⁵human embryo stem cell or induced multi-potent stem cells.

43. methods according to claim 31, wherein, the feature of the T-MSCs of described selection comprises the characteristic sum low expression level of expression CD73 further or does not express IL-6.

44. methods according to claim 31, wherein, the T-MSCs feature of described selection comprises following further: in CD90, CD105, CD13, CD29, CD54, CD146, CD166 and CD44, express at least one cell marking; Low expression level or do not express at least one cell marking being selected from CD34, CD31 and CD45; Low expression level or do not express is selected from least one mark of MMP, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α and VCAM1.

45. methods according to claim 43, wherein, the T-MSCs of described selection is by further radiation.

46. methods according to claim 44, wherein, the T-MSCs of described selection is by further radiation.

47. 1 kinds comprise the cell culture of T-MSCs selected described in claim 43.

48. 1 kinds comprise the cell culture of T-MSCs selected described in claim 44.

49. 1 kinds comprise the cell culture of T-MSCs selected described in claim 45.

50. 1 kinds comprise the cell culture of T-MSCs selected described in claim 46.

51. 1 kinds of pharmaceutical preparations, comprise the T-MSCs and pharmaceutically acceptable carrier that select described in claim 43.

52. 1 kinds of pharmaceutical preparations, comprise the T-MSCs and pharmaceutically acceptable carrier that select described in claim 44.

53. 1 kinds of pharmaceutical preparations, comprise the T-MSCs and pharmaceutically acceptable carrier that select described in claim 45.

54. 1 kinds of pharmaceutical preparations, comprise the T-MSCs and pharmaceutically acceptable carrier that select described in claim 46.

55. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 43 to improve or alleviate the symptom of disease described at least one or reverse described disease.

56. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 43 to prevent described disease progression or minimize the degree of described disease or slow down their development.

57. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 44 to improve or alleviate the symptom of disease described at least one or reverse described disease.

58. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 44 to prevent described disease progression or minimize the degree of described disease or slow down their development.

59. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 45 to improve or alleviate the symptom of disease described at least one or reverse described disease.

60. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 45 to prevent described disease progression or minimize the degree of described disease or slow down their development.

61. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 46 to improve or alleviate the symptom of disease described at least one or reverse described disease.

62. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 46 to prevent described disease progression or minimize the degree of described disease or slow down their development.

63. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 51 to improve or alleviate the symptom of disease described at least one or reverse described disease.

64. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 51 to prevent described disease progression or minimize the degree of described disease or slow down their development.

65. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 52 to improve or alleviate the symptom of disease described at least one or reverse described disease.

66. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 52 to prevent described disease progression or minimize the degree of described disease or slow down their development.

67. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 53 to improve or alleviate the symptom of disease described at least one or reverse described disease.

68. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 53 to prevent described disease progression or minimize the degree of described disease or slow down their development.

69. 1 kinds of methods for the treatment of T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 54 to improve or alleviate the symptom of disease described at least one or reverse described disease.

70. 1 kinds of methods of preventing T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 54 to prevent described disease progression or minimize the degree of described disease or slow down their development.

71. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 43 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

72. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 43 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

73. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 44 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

74. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 44 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

75. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 45 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

76. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 45 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

77. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 46 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

78. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 46 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

79. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 51 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

80. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 51 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

81. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 52 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

82. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 52 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

83. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 53 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

84. 1 kinds of methods of preventing multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 53 prevent multiple sclerosis to develop or to minimize the degree for the treatment of multiple sclerosis or the development slowing down it.

85. 1 kinds of methods for the treatment of multiple sclerosis, described method comprises to the T-MSCs having its experimenter of needs to use to select described in enough rights 54 to improve or to alleviate the symptom of at least one multiple sclerosis or to reverse multiple sclerosis.

86. 1 kinds of methods of preventing multiple sclerosis, described method comprises to there being its experimenter of needs to use the T-MSCs of the selection described in enough rights 54 to prevent multiple sclerosis to develop or minimize the degree for the treatment of multiple sclerosis or slow down its development.

Send the method for medicament through blood brain barrier and/or blood-spinal cord barrier, said method comprising the steps of for 87. 1 kinds: connect medicament to T-MSC to form T-MSC-agent complexes; And described T-MSC-agent complexes is applied to the experimenter having and need it, wherein said T-MSC can pass blood brain barrier and/or blood-spinal cord barrier and described medicament needs its experimenter to treat, to prevent, to stop or to diagnose the illness or to damage for having.

88. test kits, comprise the T-MSCs and carrier that select described in claim 43.

Test kit described in 89. claims 88, comprises melting agent, immunosuppression toughener and antihistamine.

90. methods according to claim 41, wherein, described T-MSCs gamma Rays.

91. methods according to claim 41, wherein, described T-MSCs caesium-137 gamma-radiation or carry out radiation with x-ray photon radiation.

The method of 92. 1 kinds of Therapeutic cancer or tumour, described method comprises to there being its experimenter of needs to use pharmaceutical composition described in significant quantity claim 51.

Method described in 93. claims 92, comprises further and uses the second therapeutical agent.

Method described in 94. claims 92, wherein, described experimenter used therapeutic agent treats mistake in the past.

Method described in 95. claims 92, wherein, the T-MSC of described selection by under modify in mode: genetic modification, epigenetic regulation, small molecules process, nutrition process, natural compounds or antibody treatment.

The mescenchymal stem cell (T-MSCs) of 96. trophoblastic origin modified, its method by claim 36 is produced.

97. 1 kinds of cell cultures, comprise the T-MSCs modified described in claim 96.

98. 1 kinds of pharmaceutical preparations, comprise T-MSCs and the pharmaceutically acceptable carrier modified described in claim 96.

99. 1 kinds for preventing or treat the method for T cell, B cell, inflammation and/or congenital immunity relative disease, described method comprises the T-MSCs to there being its experimenter of needs to use the modification of being produced by method described in claim 36 of significant quantity.

100. one kinds for preventing or treat the method for multiple sclerosis, described method comprises the T-MSCs to there being its experimenter of needs to use the modification of being produced by method described in claim 36 of significant quantity.

101. one kinds of test kits, comprise the T-MSCs and carrier that modify described in claim 96.

Test kit described in 102. claims 101, comprises melting agent, immunosuppression toughener and antihistamine further.

The T-MSCs modified described in 103. claims 96, wherein, the T-MSCs of described modification uses gamma Rays further.

The T-MSCs modified described in 104. claims 103, wherein, described radiation is caesium-137 gamma Rays or x-ray photon radiation.

105. methods according to claim 36, wherein, described T-MSCs is modified by with under type: shRNA, miRNA, knock out, knock in, morpholino, lure the regulation and control of RNA, DNA methylation, histone methylated regulation and control, Translational repression and/or antibody blocking, or their combination.

106. T-MSCs of described modifications produced by method described in claim 36, wherein, the feature of the T-MSCs of described modification comprises the feature expressed CD73 and low expression level or do not express IL-6 further.

The T-MSCs of the 107. described modifications of being produced by method described in claim 36, wherein, the feature of the T-MSCs of described modification comprises following characteristics further: in CD90, CD105, CD13, CD29, CD54, CD146, CD166 and CD44, at least express a kind of cell marking; Low expression level or do not express at least one cell marking being selected from CD34, CD31 and CD45; Low expression level or at least one mark of not expressing in MMP, RAGE, IFN γ R1, IFN γ R2, IL-12, TNF α and VCAM1.

108. conditioned mediums produced by method described in claim 31, the concentrated solution of conditioned medium, cell lysate or its derived prods.

109. conditioned mediums produced by method described in claim 36, the concentrated solution of conditioned medium, cell lysate or its derived prods.

110. methods according to claim 36, wherein, described MSC is the MSC in BM-MSC, T-MSC, hES-MSC or adult tissue source.

The method of 111. one kinds of Dual culture T-MSCs according to claim 43 and marrow hemopoietic stem cells and/umbilical cord hemopoietic stem cell.

Method described in 112. claims 111, wherein, described T-MSCs expresses Stro-3.

Method described in 113. claims 111, wherein, described T-MSCs expresses Stro-1.

Method described in 114. claims 111, wherein, described T-MSCs expresses Nestin.

Method described in 115. claims 111, wherein, described T-MSCs is mesenchyma stromal cells.

The coculture of 116. one kinds of T-MSCs according to claim 43 and marrow hemopoietic stem cells.

The coculture of 117. one kinds of T-MSCs according to claim 43 and umbilical cord hemopoietic stem cell.

The coculture of 118. one kinds of hES-MSC according to claim 43 and peripheral blood hematopoietic stem cells.

119. methods according to claim 31, wherein, the feature of the T-MSCs of described selection is determined by the following: flow cytometry, multiplexed microarrays, RT-PCT, RNA trace or western blotting.

120. methods according to claim 36, wherein, the feature of described T-MSCs is determined by the following: flow cytometry, multiplexed microarrays, RT-PCT, RNA trace or western blotting.

121. one kinds of mescenchymal stem cells (T-MSCs) applying trophoblastic origin carry out the method for tissue regeneration, described method to comprise to the T-MSCs having its experimenter of needs to use to select described in the claim 43-46 of significant quantity and claim 51-54 to promote tissue regeneration, includes but not limited to the regeneration of regeneration of joints, tendon, connective tissue regeneration, Neural lineage cells regeneration, adipose tissue regeneration, osteanagenesis, skin regeneration, anathrepsis, regenerating bone or cartilage, unstriated muscle regeneration, Myocardial Regeneration, epithelium regeneration and/or ligament regeneration.

Apply the method that the derivative mescenchymal stem cell (T-MSCs) of trophoderm carries out tissue repair for 122. one kinds, described method comprises to the T-MSCs having its experimenter of needs to use to select described in the claim 43-46 of significant quantity and claim 51-54 with Promotive union, include but not limited to joint healing, tendon healing, reticular tissue healing, Neural lineage cells healing, fatty tissue healing, knitting, skin healing, other wound healing, muscle healing, Cartilage healing, cardiac muscle healing, unstriated muscle healing, epithelium heals, and/or Ligament healing.

123. one kinds of methods utilizing the derivative mescenchymal stem cell (T-MSCs) of trophoderm to carry out acute tissue injury treatment, described method to comprise to the T-MSCs having its experimenter of needs to use to select described in the claim 43-46 of significant quantity and claim 51-54 to treat various acute damage, includes but not limited to that Spinal injury, acute cardiac infraction, acute radiation syndrome, Acute fire, acute fracture, acute soft tissue injury and/or acute organ are damaged.

The method of the clone that 124. one kinds of mescenchymal stem cells (T-MSCs) producing trophoblastic origin break up, described clone is at hereinafter referred to as T-MSC-DL, and described method comprises:

A described T-MSCs is taped against on the plate of gelatin, vitronectin, ln, fibronectin, matrigel or glue primordial covering by (), concentration is 1 × 10 ³cell/cm ²to 1 × 10 ⁴cells/cm ²; With

(b) serum free medium or containing the substratum of serum in cultivate T-MSCs to produce T-MSC-DL, the described substratum of serum of containing comprises bovine serum FBS or people AB serum ABHS.

The method of the clone that 125. one kinds of mescenchymal stem cells (T-MSCs) producing trophoblastic origin break up, described clone is at hereinafter referred to as T-MSC-DL, described method comprises: cultivate the described T-MSC mono-suitable period in the medium to produce T-MSC-DL, described substratum comprises 1-50ng/ml, preferred 10ng/ml, FGF2 (FGF-2); 1-50ng/ml, preferred 10ng/m, Urogastron; And 0.5-5ng/ml, preferred 1ng/ml, platelet derived growth factor (PDGF).

The method of the dopaminergic neuron phenotype cells system that 126. one kinds of mescenchymal stem cells (T-MSCs) producing trophoblastic origin break up, said method comprising the steps of:

A () is cultivated the described T-MSCs mono-suitable period in the medium and is broken up towards neural destiny to make T-MSCs, described substratum comprises 1-50ng/ml, preferred 10ng/ml, FGF2 (FGF-2); 1-50ng/ml, preferred 10ng/m, Urogastron;

B the T-MSCs of () culturing step (a) is in the medium to cause midbrain specialization, described substratum comprises 10-200ng/ml, preferred 100ng/ml, Sonic hedgehog (SHH); 1-50ng/ml, preferred 10ng/ml, FGF-8 (people); With 50-500 μM, preferably 200 μMs, APP;

C the T-MSCs of () culturing step (b) is in the medium to induce T-MSCs to dopaminergic neuron phenotype cells system differentiation and ripe, described substratum comprises 5-500ng/ml, preferred 50ng/ml, the glial-derived neurotrophic factor (GDNF) and 50-500 μM, preferably 200 μMs, APP.

Produce the method for Osteogenesis phenotype clone of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 127. one kinds, described method comprises cultivates the described T-MSCs mono-suitable period in the medium to induce T-MSCs to osteoblast differentiation, used medium comprises low dextrose DMEM, adds 10%FCS; 1-150 μM, preferably 80 μMs, AA-2P; 0.5-5 μM, preferably 1 μM, dexamethasone; And 1-100mM, preferred 20mM, beta-glycerophosphate.

Produce the method for lipogenesis phenotype cells system of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 128. one kinds, described method comprises cultivates the described T-MSCs mono-suitable period in the medium and breaks up to elaioplast to induce T-MSCs, used medium comprises low dextrose DMEM, adds 20%FCS; 1-10 μ g/ml, preferably 5 μ g/ml, Regular Insulin; 0.5-10 μM, preferably 2 μMs, dexamethasone; 0.1-1mM, preferred 0.5mM, isobutyl methylxanthine; With 1-100 μM, preferably 60 μMs, indomethacin.

Produce the method for Subchondral drilling phenotype cells system of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 129. one kinds, described method comprises cultivates the described T-MSCs mono-suitable period in the medium in pellet form and breaks up to chondroblast to induce T-MSCs, described substratum comprises high glucose sugar DMEM, add 0.5-10mM, preferred 1mM, Sodium.alpha.-ketopropionate; 0.5-1mM, preferred 0.1mM, AA-2P; 0.05-1 μM, preferably 0.1 μM, dexamethasone; 0.2-2%, preferably 1%, ITS; And 1-50ng/ml, preferred 10ng/ml, TGF-β 3.

Produce the method for myogenicity phenotype cells system of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin, said method comprising the steps of for 130. one kinds:

A () cultivates the described T-MSCs mono-suitable period in the medium, described substratum comprises low dextrose DMEM, adds 10%FBS; 1-20 μM, preferably 10 μMs, 5-azacytidine; And 1-50ng/ml, preferred 10ng/ml, basic FGF, 24 hours;

D () adds 10%FBS with comprising DMEM; 1-50ng/ml, preferred 10ng/ml, the substratum of substratum exchonge step (a) of basic FGF, to induce T-MSCs myogenic differentiation.

Produce the method for inoblast phenotype cells system of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 131. one kinds, described method comprises cultivates the described T-MSCs mono-suitable period in the medium and breaks up to induce T-MSCs inoblast, and described substratum comprises DMEM and adds 10%FBS; 50-200ng/ml, preferred 100ng/ml, Connective Tissue Growth Factor (CTGF); With 1-100 μ g/ml, preferably 50 μ g/ml, xitix.

Produce the method for clone of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 132. one kinds, described clone, at hereinafter referred to as T-MSC-DL, said method comprising the steps of:

A () cultivates the described T-MSCs mono-suitable period in the medium, described substratum comprises Neurobasal medium, with the addition of 0.25x B-27 fill-in; 10-200ng/ml, preferred 100ng/ml, Sonic hedgehog (SHH); 1-50ng/ml, preferred 10ng/ml, FGF-8 (mouse), and 1-200ng/ml, preferred 50ng/ml, FGF-2; With

B () gathers in the crops described T-MSC-DL after step (a) cultivates 6 to 12 days.

Produce the method for clone of being broken up by the mescenchymal stem cell (T-MSCs) of trophoblastic origin for 133. one kinds, described clone, at hereinafter referred to as T-MSC-DL, said method comprising the steps of:

A () cultivates described T-MSCs 48-72 hour in the medium, described substratum comprises serum free medium and adds 2mM glutamine; 1-20U/ml, preferred 12.5U/ml, nystatin; N2 fill-in; 2-50ng/ml, preferred 20ng/ml, FGF2 (FGF-2); 1-50ng/mL, preferred 10ng/ml, EGF; With

B () the described T-MSCs of culturing step (a) is to produce T-MSC-DL in the medium, described substratum comprises Neurobasal medium, adds B27 fill-in; 0.1-10mM, preferred 1mM, two butyryl cyclisation AMP (dbcAMP); 3-isobutyl-1-methylxanthine (IBMX); 10-500 μM, preferably 200 μMs, xitix; 1-100ng/ml, preferred 50ng/ml, BDNF; 1-50ng/ml, preferred 10ng/ml, the glial-derived neurotrophic factor (GDNF); 0.2-10ng/ml, preferred 2ng/ml, transforming growth factor-beta 3 (TGF-β 3) and 0.05-5 Μ m, preferably 0.1 μM, all-trans-retinoic acid (RA).